Spiro-lactam nmda receptor modulators and uses thereof

ABSTRACT

Disclosed are compounds having enhanced potency in the modulation of NMDA receptor activity. Such compounds are contemplated for use in the treatment of conditions such as depression and related disorders. Orally available formulations and other pharmaceutically acceptable delivery forms of the compounds, including intravenous formulations, are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/757,903, filed on Jan. 29, 2013, which is incorporated by referencein its entirety.

BACKGROUND

An N-methyl-d-aspartate (NMDA) receptor is a postsynaptic, ionotropicreceptor that is responsive to, inter alia, the excitatory amino acidsglutamate and glycine and the synthetic compound NMDA. The NMDA receptorcontrols the flow of both divalent and monovalent ions into thepostsynaptic neural cell through a receptor associated channel (Fosteret al., Nature 1987, 329:395-396; Mayer et al., Trends in Pharmacol.Sci. 1990, 11:254-260). The NMDA receptor has been implicated duringdevelopment in specifying neuronal architecture and synapticconnectivity, and may be involved in experience-dependent synapticmodifications. In addition, NMDA receptors are also thought to beinvolved in long term potentiation and central nervous system disorders.

The NMDA receptor plays a major role in the synaptic plasticity thatunderlies many higher cognitive functions, such as memory acquisition,retention and learning, as well as in certain cognitive pathways and inthe perception of pain (Collingridge et al., The NMDA Receptor, OxfordUniversity Press, 1994). In addition, certain properties of NMDAreceptors suggest that they may be involved in theinformation-processing in the brain that underlies consciousness itself.

The NMDA receptor has drawn particular interest since it appears to beinvolved in a broad spectrum of CNS disorders. For instance, duringbrain ischemia caused by stroke or traumatic injury, excessive amountsof the excitatory amino acid glutamate are released from damaged oroxygen deprived neurons. This excess glutamate binds to the NMDAreceptors which opens their ligand-gated ion channels; in turn thecalcium influx produces a high level of intracellular calcium whichactivates a biochemical cascade resulting in protein degradation andcell death. This phenomenon, known as excitotoxicity, is also thought tobe responsible for the neurological damage associated with otherdisorders ranging from hypoglycemia and cardiac arrest to epilepsy. Inaddition, there are preliminary reports indicating similar involvementin the chronic neurodegeneration of Huntington's, Parkinson's, andAlzheimer's diseases. Activation of the NMDA receptor has been shown tobe responsible for post-stroke convulsions, and, in certain models ofepilepsy, activation of the NMDA receptor has been shown to be necessaryfor the generation of seizures. Neuropsychiatric involvement of the NMDAreceptor has also been recognized since blockage of the NMDA receptorCa⁺⁺ channel by the animal anesthetic PCP (phencyclidine) produces apsychotic state in humans similar to schizophrenia (reviewed in Johnson,K. and Jones, S., 1990). Further, NMDA receptors have also beenimplicated in certain types of spatial learning.

The NMDA receptor is believed to consist of several protein chainsembedded in the postsynaptic membrane. The first two types of subunitsdiscovered so far form a large extracellular region, which probablycontains most of the allosteric binding sites, several transmembraneregions looped and folded so as to form a pore or channel, which ispermeable to Ca⁺⁺, and a carboxyl terminal region. The opening andclosing of the channel is regulated by the binding of various ligands todomains (allosteric sites) of the protein residing on the extracellularsurface. The binding of the ligands is thought to affect aconformational change in the overall structure of the protein which isultimately reflected in the channel opening, partially opening,partially closing, or closing.

NMDA receptor compounds may exert dual (agonist/antagonist) effect onthe NMDA receptor through the allosteric sites. These compounds aretypically termed “partial agonists”. In the presence of the principalsite ligand, a partial agonist will displace some of the ligand and thusdecrease Ca⁺⁺ flow through the receptor. In the absence of or loweredlevel of the principal site ligand, the partial agonist acts to increaseCa⁺⁺ flow through the receptor channel.

A need continues to exist in the art for novel and more specific/potentcompounds that are capable of binding the glycine binding site of NMDAreceptors, and provide pharmaceutical benefits. In addition, a needcontinues to exist in the medical arts for orally deliverable forms ofsuch compounds.

SUMMARY

Provided herein, at least in part, are compounds that are NMDAmodulators, for example, partial agonists of NMDA. For example,disclosed herein are compounds represented by the formula:

and pharmaceutically acceptable salts, stereoisomers, and N-oxidesthereof, wherein

-   -   R_(b) is selected from the group consisting of H, halogen,        hydroxyl, cyano and C₁-C₆ alkyl;    -   R₁ is H or C₁-C₆ alkyl;    -   R₂ is H or C₁-C₆ alkyl;    -   R₃ is selected from the group consisting of H, C₁-C₆ alkyl and a        nitrogen protecting group;    -   R₄ and R₅ are independently H or C₁-C₆ alkyl, or R₄ and R₅ taken        together with the nitrogen to which they are attached form a 4-,        5- or 6-membered heterocyclic or heteroaryl ring optionally        substituted with one or more substituents selected from the        group consisting of halogen, cyano, oxo, C₁-C₆ alkyl, —OH, C₁-C₆        alkoxy, and —N(R′)R′, wherein R′ is independently selected for        each occurrence from H or C₁-C₆ alkyl;    -   R₆ is selected from the group consisting of —OH, C₁-C₆ alkoxy,        —OC(O)—C₁-C₆ alkyl, and —OC(O)phenyl; and    -   R₇ is H or C₁-C₆ alkyl;        or in other embodiments, the variables set forth in formula (I)        are defined as follows:    -   R_(b) is selected from the group consisting of H, halogen,        hydroxyl, cyano and C₁-C₆ alkyl (e.g., H);    -   R₁ is H or C₁-C₆ alkyl;    -   R₂ is H or C₁-C₆ alkyl;    -   R₃ is selected from the group consisting of H, C₁-C₆ alkyl and a        nitrogen protecting group;    -   R₄ and R₅ are each independently selected from the group        consisting of H, C₁-C₆ alkyl,    -   X, and —C₁-C₆ alkylene-X, wherein X is selected from the group        consisting of:        -   (i) C₃-C₆ cycloalkyl;        -   (ii) heteroaryl including from 5 to 6 ring atoms wherein 1,            2, or 3 of the ring atoms are independently selected from            the group consisting of N, NH, N(C1-C3 alkyl), O, and S;        -   (iii) heterocyclyl including from 3 to 6 ring atoms wherein            1, 2, or 3 of the ring atoms are independently selected from            the group consisting of N, NH, N(C1-C3 alkyl), O, and S; and        -   (iv) phenyl;    -   wherein C₃-C₆ cycloalkyl and heterocyclyl are each optionally        substituted with from 1-3 substituents independently selected        from the group consisting of halogen, cyano, oxo, C₁-C₆ alkyl,        hydroxyl, C₁-C₆ alkoxy, and —N(R′)R′, wherein R′ is        independently selected for each occurrence from H and C₁-C₆        alkyl; and heteroaryl and phenyl are each optionally substituted        with from 1-3 substituents independently selected from the group        consisting of halogen, cyano, C₁-C₆ alkyl, hydroxyl, C₁-C₆        alkoxy, and —N(R′)R′;    -   or R₄ and R₅ together with the nitrogen to which they are        attached form:        -   heterocyclyl including from 4 to 6 ring atoms; wherein the            heterocyclyl includes not more than two ring heteroatoms            (including the nitrogen atom attached to R₄ and R₅), and the            second ring heteroatom, when present, is independently            selected from the group consisting of N, NH, N(C1-C3 alkyl),            O, and S; and wherein the heterocyclyl is optionally            substituted with from 1-3 substituents independently            selected from the group consisting of halogen, cyano, oxo,            C₁-C₆ alkyl, hydroxyl, C₁-C₆ alkoxy, and —N(R′)R′; or        -   heteroaryl including from 5 to 6 ring atoms; wherein the            heteroaryl includes not more than four ring heteroatoms            (including the nitrogen atom attached to R₄ and R₅), and            each additional ring heteroatom, when present, is            independently selected from the group consisting of N, NH,            N(C1-C3 alkyl), O, and S; and wherein the heteroaryl is            optionally substituted with from 1-3 substituents            independently selected from the group consisting of halogen,            cyano, C₁-C₆ alkyl, hydroxyl, C₁-C₆ alkoxy, and —N(R′)R′;    -   R₆ is selected from the group consisting of —OH, C₁-C₆ alkoxy,        —OC(O)—C₁-C₆ alkyl, —OC(O)phenyl, and —N(R′)R′; and    -   R₇ is H or C₁-C₆ alkyl.

Also provided herein are pharmaceutically acceptable compositionscomprising a disclosed compound, and a pharmaceutically acceptableexcipient. For example, such compositions may be suitable for oral orintravenous administration to a patient.

In another aspect, a method of treating a condition selected from thegroup consisting of autism, anxiety, depression, bipolar disorder,attention deficit disorder, attention deficit hyperactivity disorder(ADHD), schizophrenia, a psychotic disorder, a psychotic symptom, socialwithdrawal, obsessive-compulsive disorder, phobia, post-traumatic stresssyndrome, a behavior disorder, an impulse control disorder, a substanceabuse disorder, a sleep disorder, a memory disorder, a learningdisorder, urinary incontinence, multiple system atrophy, progressivesupra-nuclear palsy, Friedrich's ataxia, Down's syndrome, fragile Xsyndrome, tuberous sclerosis, olivio-ponto-cerebellar atrophy, cerebralpalsy, drug-induced optic neuritis, ischemic retinopathy, diabeticretinopathy, glaucoma, dementia, AIDS dementia, Alzheimer's disease,Huntington's chorea, spasticity, myoclonus, muscle spasm, Tourette'ssyndrome, epilepsy, cerebral ischemia, stroke, a brain tumor, traumaticbrain injury, cardiac arrest, myelopathy, spinal cord injury, peripheralneuropathy, acute neuropathic pain, and chronic neuropathic, in apatient in need thereof is provided. Such methods may compriseadministering to the patient a pharmaceutically effective amount of adisclosed compound or pharmaceutically acceptable salts, stereoisomers,N-oxides, and hydrates thereof.

In some embodiments, a contemplated method includes treating depression.For example, depression may include one or more of major depressivedisorder, dysthymic disorder, psychotic depression, postpartumdepression, seasonal affective disorder, bipolar disorder, mooddisorder, or depression caused by a chronic medical condition. In otherembodiments, a contemplated method may treat schizophrenia. Suchschizophrenia may be, for example, paranoid type schizophrenia,disorganized type schizophrenia, catatonic type schizophrenia,undifferentiated type schizophrenia, residual type schizophrenia,post-schizophrenic depression, or simple schizophrenia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the potentiation of [³H]MK-801 binding in the presence ofCompound X.

FIG. 2 shows results of long term potentiation in hippocampal slicesusing Compound X.

DETAILED DESCRIPTION

This disclosure is generally directed to compounds that are capable ofmodulating NMDA, e.g., NMDA antagonists or partial agonists, andcompositions and/or methods of using the disclosed compounds.

Definitions

“Treating” includes any effect, e.g., lessening, reducing, modulating,or eliminating, that results in the improvement of the condition,disease, disorder and the like.

The term “alkenyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon double bond, suchas a straight or branched group of 2-6 or 3-4 carbon atoms, referred toherein for example as C₂-C₆ alkenyl, and C₃-C₄ alkenyl, respectively.Exemplary alkenyl groups include, but are not limited to, vinyl, allyl,butenyl, pentenyl, etc.

The term “alkoxy” as used herein refers to a straight or branched alkylgroup attached to an oxygen (alkyl-O—). Exemplary alkoxy groups include,but are not limited to, alkoxys of 1-6 or 2-6 carbon atoms, referred toherein as C₁-C₆ alkoxy, and C₂-C₆ alkoxy, respectively. Exemplary alkoxygroups include, but are not limited to methoxy, ethoxy, isopropoxy, etc.

The term “alkenyloxy” used herein refers to a straight or branchedalkenyl group attached to an oxygen (alkenyl-O). Exemplary alkenoxygroups include, but are not limited to, groups with an alkenyl group of3-6 carbon atoms, (also e.g. referred to as C₃-C₆ alkenyloxy). Exemplary“alkenoxy” groups include, but are not limited to allyloxy, butenyloxy,etc.

The term “alkynyloxy” used herein refers to a straight or branchedalkynyl group attached to an oxygen (alkynyl-O)). Exemplary alkynyloxygroups include, but are not limited to, C₃-C₆ alkynyloxy, e.g.,propynyloxy.

The term “alkyl” as used herein refers to a saturated straight orbranched hydrocarbon, such as a straight or branched group of 1-6, 1-4,or 1-3 carbon atoms, referred to herein as C₁-C₆ alkyl, C₁-C₄ alkyl, andC₁-C₃ alkyl, respectively. Exemplary alkyl groups include, but are notlimited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl,2-methyl-2-propyl, 2-methyl-1-butyl, 3 methyl 1 butyl 3-methyl-2-butyl,2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl,4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl,2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl,hexyl, etc. The term “haloalkyl” as used herein refers to a saturatedstraight or branched alkyl groups, in which one or more hydrogen atomsof the alkyl group are replaced with one or more independently selectedhalogens. The term “haloalkyl” encompasses alkyl groups in which all ofhydrogen atoms of the alkyl group are replaced independently selectedhalogens (sometimes referred to as “perhalo” alkyl groups. Exemplaryhaloalkyl groups include, but are not limited to, CH₂F, CH₂CH₂Cl, CF₃,CHFCH₂Cl.

The term “alkynyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon triple bond, suchas a straight or branched group of 2-6, or 3-6 carbon atoms, referred toherein as C₂-C₆ alkynyl, and C₃-C₆alkynyl, respectively. Exemplaryalkynyl groups include, but are not limited to, ethynyl, propynyl,butynyl, pentynyl, hexynyl, methylpropynyl, etc.

The term “bridged cycloalkyl”, as used herein, is defined as amonocyclic 4- to 7-membered cycloalkyl group in which two non-adjacentatoms are linked by a CH₂ or CH₂CH₂ group. A “bridged cycloalkyl” may befused to one or more phenyl, partially unsaturated, or saturated rings.Examples of bridged carbocyclic groups include but are not limited tobicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[2.2.2]octene etc.

The term “carbonyl” as used herein refers to the radical —C(O)—. Theterm “cyano” as used herein refers to the radical —CN. The term “nitro”refers to the radical —NO₂. The term “H” refers to hydrogen.

The term “cycloalkoxy” as used herein refers to a cycloalkyl groupattached to an oxygen (cycloalkyl-O—).

The term “cycloalkyl” as used herein refers to a monocyclic saturated orpartically unsatured hydrocarbon group of for example 3-6, or 4-6carbons, referred to herein, e.g., as “C₃₋₆ cycloalkyl” or “C₄₋₆cycloalkyl,” and derived from a cycloalkane. Exemplary cycloalkyl groupsinclude, but are not limited to, cyclohexane, cyclohexene, cyclopentane,cyclobutane, cyclopropane or cyclopentane.

The terms “halo” or “halogen” as used herein refer to F, Cl, Br, or I.

The terms “heteroaryl” as used herein refers to a monocyclic aromatic4-6 membered ring system containing one or more heteroatoms, for exampleone to three heteroatoms, such as nitrogen, oxygen, and sulfur. Wherepossible, said heteroaryl ring may be linked to the adjacent radicalthough carbon or nitrogen. Examples of heteroaryl rings include but arenot limited to furan, thiophene, pyrrole, thiazole, oxazole,isothiazole, isoxazole, imidazole, pyrazole, triazole, pyridine, andpyrimidine.

The terms “heterocyclyl” or “heterocyclic group” are art-recognized andrefer to saturated or partially unsaturated 4- to 7-membered ringstructures, whose ring structures include one to three heteroatoms, suchas nitrogen, oxygen, and sulfur. A heterocycle may be fused to one ormore phenyl, partially unsaturated, or saturated rings. Examples ofheterocyclyl groups include but are not limited to pyrrolidine,piperidine, morpholine, thiomorpholine, and piperazine.

The term “heterocyclylalkoxy” as used herein refers to aheterocyclyl-alkyl-O— group.

The term “heterocyclyloxyalkyl” refers to a heterocyclyl-O-alkyl- group.

The term “heterocycloxy” refers to a heterocyclyl-O— group. The term“cycloalkyloxy” refers to a cycloalkyl-O— group.

The term “heteroaryloxy” referes to a heteroaryl-O— group.

The terms “hydroxy” and “hydroxyl” as used herein refers to the radical—OH.

The term “oxo” as used herein refers to the radical ═O.

The term “nitrogen protecting group” or “amino protecting group” isart-recognized and as used herein refers to a chemical moiety that iscovalently linked to a nitrogen atom of an amino (primary or secondary)group and that temporarily blocks the reactivity of the amino groupduring a synthetic step and is selectively removed once the syntheticstep is complete. Nitrogen protecting groups include, for example,9-Fluorenylmethyloxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc),carbobenzyloxycarbonyl (Cbz), p-methoxybenzyloxycarbonyl, acetyl,trifluoroacetyl, benzoyl, phthalimido, benzyl (Bn), p-methoxybenzyl,p-methoxyphenyl, 3,4-dimethoxybenzyl, triphenylmethyl, benzylidene, andp-toluenesulfonyl (Ts). In some embodiments, the nitrogen protectinggroup can have one of the following formulas: —C(O)OR₃₁ or —C(O)R₃₂ asdefined herein.

As used in the present disclosure, the term “partial NMDA receptoragonist” generally refers to a compound that is capable of binding to aglycine binding site of an NMDA receptor; at low concentrations a NMDAreceptor agonist acts substantially as agonist and at highconcentrations it acts substantially as an antagonist. Theseconcentrations are experimentally determined for each and every “partialagonist.

“Pharmaceutically or pharmacologically acceptable” include molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate. For human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” as used herein refers to any and all solvents,dispersion media, coatings, isotonic and absorption delaying agents, andthe like, that are compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. The compositions may also contain other activecompounds providing supplemental, additional, or enhanced therapeuticfunctions.

The term “pharmaceutical composition” as used herein refers to acomposition comprising at least one compound as disclosed hereinformulated together with one or more pharmaceutically acceptablecarriers.

“Individual,” “patient,” or “subject” are used interchangeably andinclude any animal, including mammals, preferably mice, rats, otherrodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates,and most preferably humans. The compounds of the invention can beadministered to a mammal, such as a human, but can also be administeredto other mammals such as an animal in need of veterinary treatment,e.g., domestic animals (e.g., dogs, cats, and the like), farm animals(e.g., cows, sheep, pigs, horses, and the like) and laboratory animals(e.g., rats, mice, guinea pigs, and the like). The mammal treated in themethods of the invention is desirably a mammal in which treatment e.g.,of pain or depression is desired. “Modulation” includes antagonism(e.g., inhibition), agonism, partial antagonism and/or partial agonism.

In the present specification, the term “therapeutically effectiveamount” means the amount of the subject compound that will elicit thebiological or medical response of a tissue, system, animal or human thatis being sought by the researcher, veterinarian, medical doctor or otherclinician. The compounds of the invention are administered intherapeutically effective amounts to treat a disease. Alternatively, atherapeutically effective amount of a compound is the quantity requiredto achieve a desired therapeutic and/or prophylactic effect, such as anamount which results in lessening a symptom of depression.

The term “pharmaceutically acceptable salt(s)” as used herein refers tosalts of acidic or basic groups that may be present in compounds used inthe present compositions. Compounds included in the present compositionsthat are basic in nature are capable of forming a wide variety of saltswith various inorganic and organic acids. The acids that may be used toprepare pharmaceutically acceptable acid addition salts of such basiccompounds are those that form non-toxic acid addition salts, i.e., saltscontaining pharmacologically acceptable anions, including but notlimited to malate, oxalate, chloride, bromide, iodide, nitrate, sulfate,bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate,salicylate, citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.Compounds included in the present compositions that are acidic in natureare capable of forming base salts with various pharmacologicallyacceptable cations. Examples of such salts include alkali metal oralkaline earth metal salts and, particularly, calcium, magnesium,sodium, lithium, zinc, potassium, and iron salts. Compounds included inthe present compositions that include a basic or acidic moiety may alsoform pharmaceutically acceptable salts with various amino acids. Thecompounds of the disclosure may contain both acidic and basic groups;for example, one amino and one carboxylic acid group. In such a case,the compound can exist as an acid addition salt, a zwitterion, or a basesalt.

The compounds of the disclosure may contain one or more chiral centersand/or double bonds and, therefore, exist as stereoisomers, such asgeometric isomers, enantiomers or diastereomers. The term“stereoisomers” when used herein consist of all geometric isomers,enantiomers or diastereomers. These compounds may be designated by thesymbols “R” or “S,” depending on the configuration of substituentsaround the stereogenic carbon atom. The present invention encompassesvarious stereoisomers of these compounds and mixtures thereof.Stereoisomers include enantiomers and diastereomers. Mixtures ofenantiomers or diastereomers may be designated “(±)” in nomenclature,but the skilled artisan will recognize that a structure may denote achiral center implicitly.

The compounds of the disclosure may contain one or more chiral centersand/or double bonds and, therefore, exist as geometric isomers,enantiomers or diastereomers. The enantiomer and diastereomers may bedesignated by the symbols “(±),” “(−).” “R” or “S,” depending on theconfiguration of substituents around the stereogenic carbon atom, butthe skilled artisan will recognize that a structure may denote a chiralcenter implicitly. Geometric isomers, resulting from the arrangement ofsubstituents around a carbon-carbon double bond or arrangement ofsubstituents around a cycloalkyl or heterocyclic ring, can also exist inthe compounds of the present invention. The symbol

denotes a bond that may be a single, double or triple bond as describedherein. Substituents around a carbon-carbon double bond are designatedas being in the “Z” or “E” configuration wherein the terms “Z” and “E”are used in accordance with IUPAC standards. Unless otherwise specified,structures depicting double bonds encompass both the “E” and “Z”isomers. Substituents around a carbon-carbon double bond alternativelycan be referred to as “cis” or “trans,” where “cis” representssubstituents on the same side of the double bond and “trans” representssubstituents on opposite sides of the double bond. The arrangement ofsubstituents around a carbocyclic ring can also be designated as “cis”or “trans.” The term “cis” represents substituents on the same side ofthe plane of the ring and the term “trans” represents substituents onopposite sides of the plane of the ring. Mixtures of compounds whereinthe substituents are disposed on both the same and opposite sides ofplane of the ring are designated “cis/trans.”

The term “stereoisomers” when used herein consist of all geometricisomers, enantiomers or diastereomers. The present invention encompassesvarious stereoisomers of these compounds and mixtures thereof.

Individual enantiomers and diasteriomers of compounds of the presentinvention can be prepared synthetically from commercially availablestarting materials that contain asymmetric or stereogenic centers, or bypreparation of racemic mixtures followed by resolution methods wellknown to those of ordinary skill in the art. These methods of resolutionare exemplified by (1) attachment of a mixture of enantiomers to achiral auxiliary, separation of the resulting mixture of diastereomersby recrystallization or chromatography and liberation of the opticallypure product from the auxiliary, (2) salt formation employing anoptically active resolving agent, (3) direct separation of the mixtureof optical enantiomers on chiral liquid chromatographic columns or (4)kinetic resolution using steroselective chemical or enzymatic reagents.Racemic mixtures can also be resolved into their component enantiomersby well-known methods, such as chiral-phase gas chromatography orcrystallizing the compound in a chiral solvent. Stereoselectivesyntheses, a chemical or enzymatic reaction in which a single reactantforms an unequal mixture of stereoisomers during the creation of a newstereocenter or during the transformation of a pre-existing one, arewell known in the art. Stereoselective syntheses encompass both enantio-and diastereoselective transformations. For examples, see Carreira andKvaerno, Classics in Stereoselective Synthesis, Wiley-VCH: Weinheim,2009.

The compounds disclosed herein can exist in solvated as well asunsolvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms. In one embodiment, thecompound is amorphous. In one embodiment, the compound is a singlepolymorph. In another embodiment, the compound is a mixture ofpolymorphs. In another embodiment, the compound is in a crystallineform.

The invention also embraces isotopically labeled compounds of theinvention which are identical to those recited herein, except that oneor more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. Examples of isotopes that can be incorporated into compounds ofthe invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorus, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O,¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively. For example, a compoundof the invention may have one or more H atom replaced with deuterium.

Certain isotopically-labeled disclosed compounds (e.g., those labeledwith ³H and ¹⁴C) are useful in compound and/or substrate tissuedistribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C)isotopes are particularly preferred for their ease of preparation anddetectability. Further, substitution with heavier isotopes such asdeuterium (i.e., ²H) may afford certain therapeutic advantages resultingfrom greater metabolic stability (e.g., increased in vivo half-life orreduced dosage requirements) and hence may be preferred in somecircumstances. Isotopically labeled compounds of the invention cangenerally be prepared by following procedures analogous to thosedisclosed in the e.g., Examples herein by substituting an isotopicallylabeled reagent for a non-isotopically labeled reagent.

The term “prodrug” refers to compounds that are transformed in vivo toyield a disclosed compound or a pharmaceutically acceptable salt,hydrate or solvate of the compound. The transformation may occur byvarious mechanisms (such as by esterase, amidase, phosphatase, oxidativeand or reductive metabolism) in various locations (such as in theintestinal lumen or upon transit of the intestine, blood or liver).Prodrugs are well known in the art (for example, see Rautio,Kumpulainen, et al, Nature Reviews Drug Discovery 2008, 7, 255). Forexample, if a compound of the invention or a pharmaceutically acceptablesalt, hydrate or solvate of the compound contains a carboxylic acidfunctional group, a prodrug can comprise an ester formed by thereplacement of the hydrogen atom of the acid group with a group such as(C₁-C₈)alkyl, (C₂-C₁₂)alkanoyloxymethyl, 1-(alkanoyloxy)ethyl havingfrom 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having from 5to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbonatoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms,1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms,N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms,1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms,3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl,di-N,N—(C₁-C₂)alkylamino(C₂-C₃)alkyl (such as β-dimethylaminoethyl),carbamoyl-(C₁-C₂)alkyl, N,N-di(C₁-C₂)alkylcarbamoyl-(C₁-C₂)alkyl andpiperidino-, pyrrolidino- or morpholino(C₂-C₁)alkyl.

Similarly, if a compound of the invention contains an alcohol functionalgroup, a prodrug can be formed by the replacement of the hydrogen atomof the alcohol group with a group such as (C₁-C₆)alkanoyloxymethyl,1-((C₁-C₆)alkanoyloxy)ethyl, 1-methyl-1-((C₁-C₆)alkanoyloxy)ethyl(C₁-C₆)alkoxycarbonyloxymethyl, N—(C₁-C₆)alkoxycarbonylaminomethyl,succinoyl, (C₁-C₆)alkanoyl, α-amino(C₁-C₄)alkanoyl, arylacyl andα-aminoacyl, or α-aminoacyl-α-aminoacyl, where each α-aminoacyl group isindependently selected from the naturally occurring L-amino acids,P(O)(OH)₂, —P(O)(O(C₁-C₆)alkyl)₂ or glycosyl (the radical resulting fromthe removal of a hydroxyl group of the hemiacetal form of acarbohydrate).

If a compound of the invention incorporates an amine functional group, aprodrug can be formed, for example, by creation of an amide orcarbamate, an N-acyloxyakyl derivative, an (oxodioxolenyl)methylderivative, an N-Mannich base, imine or enamine. In addition, asecondary amine can be metabolically cleaved to generate a bioactiveprimary amine, or a tertiary amine can metabolically cleaved to generatea bioactive primary or secondary amine. For examples, see Simplicio, etal., Molecules 2008, 13, 519 and references therein.

Compounds

Disclosed compounds include those represented by the formula:

and pharmaceutically acceptable salts, stereoisomers, and N-oxidesthereof, wherein

-   -   R_(b) is selected from the group consisting of H, halogen,        hydroxyl, cyano and C₁-C₆ alkyl;    -   R₁ is H or C₁-C₆ alkyl;    -   R₂ is H or C₁-C₆ alkyl;    -   R₃ is selected from the group consisting of H, C₁-C₆ alkyl and a        nitrogen protecting group;    -   R₄ and R₅ are independently H or C₁-C₆ alkyl, or R₄ and R₅ taken        together with the nitrogen to which they are attached form a 4-,        5- or 6-membered heterocyclic or heteroaryl ring optionally        substituted with one or more substituents selected from the        group consisting of halogen, cyano, oxo, C₁-C₆ alkyl, —OH, C₁-C₆        alkoxy, and —N(R′)R′, wherein R′ is independently selected for        each occurrence from H or C₁-C₆ alkyl;    -   R₆ is selected from the group consisting of —OH, C₁-C₆ alkoxy,        —OC(O)—C₁-C₆ alkyl, and —OC(O)phenyl; and    -   R₇ is H or C₁-C₆ alkyl;        or in other embodiments, the variables set forth in formula (I)        are defined as follows:    -   R_(b) is selected from the group consisting of H, halogen,        hydroxyl, cyano and C₁-C₆ alkyl (e.g., H);    -   R₁ is H or C₁-C₆ alkyl;    -   R₂ is H or C₁-C₆ alkyl;    -   R₃ is selected from the group consisting of H, C₁-C₆ alkyl and a        nitrogen protecting group;    -   R₄ and R₅ are each independently selected from the group        consisting of H, C₁-C₆ alkyl,    -   X, and —C₁-C₆ alkylene-X, wherein X is selected from the group        consisting of:        -   (i) C₃-C₆ cycloalkyl;        -   (ii) heteroaryl including from 5 to 6 ring atoms wherein 1,            2, or 3 of the ring atoms are independently selected from            the group consisting of N, NH, N(C1-C3 alkyl), O, and S;        -   (iii) heterocyclyl including from 3 to 6 ring atoms wherein            1, 2, or 3 of the ring atoms are independently selected from            the group consisting of N, NH, N(C1-C3 alkyl), O, and S; and        -   (iv) phenyl;    -   wherein C₃-C₆ cycloalkyl and heterocyclyl are each optionally        substituted with from 1-3 substituents independently selected        from the group consisting of halogen, cyano, oxo, C₁-C₆ alkyl,        hydroxyl, C₁-C₆ alkoxy, and —N(R′)R′, wherein R′ is        independently selected for each occurrence from H and C₁-C₆        alkyl; and heteroaryl and phenyl are each optionally substituted        with from 1-3 substituents independently selected from the group        consisting of halogen, cyano, C₁-C₆ alkyl, hydroxyl, C₁-C₆        alkoxy, and —N(R′)R′;    -   or R₄ and R₅ together with the nitrogen to which they are        attached form:        -   heterocyclyl including from 4 to 6 ring atoms; wherein the            heterocyclyl includes not more than two ring heteroatoms            (including the nitrogen atom attached to R₄ and R₅), and the            second ring heteroatom, when present, is independently            selected from the group consisting of N, NH, N(C1-C3 alkyl),            O, and S; and wherein the heterocyclyl is optionally            substituted with from 1-3 substituents independently            selected from the group consisting of halogen, cyano, oxo,            C₁-C₆ alkyl, hydroxyl, C₁-C₆ alkoxy, and —N(R′)R′; or        -   heteroaryl including from 5 to 6 ring atoms; wherein the            heteroaryl includes not more than four ring heteroatoms            (including the nitrogen atom attached to R₄ and R₅), and            each additional ring heteroatom, when present, is            independently selected from the group consisting of N, NH,            N(C1-C3 alkyl), O, and S; and wherein the heteroaryl is            optionally substituted with from 1-3 substituents            independently selected from the group consisting of halogen,            cyano, C₁-C₆ alkyl, hydroxyl, C₁-C₆ alkoxy, and —N(R′)R′;    -   R₆ is selected from the group consisting of —OH, C₁-C₆ alkoxy,        —OC(O)—C₁-C₆ alkyl, —OC(O)phenyl, and —N(R′)R′; and    -   R₇ is H or C₁-C₆ alkyl.

In some embodiments, R₁ is H. In other embodiments, R₁ is C₁-C₆ alkyl,e.g., CH₃.

In some embodiments, R₂ is H. In other embodiments, R₂ is C₁-C₆ alkyl,e.g., CH₃.

In some embodiments, R₃ is H.

In some embodiments, R₃ is a nitrogen protecting group. In someembodiments, R₃ has formula —C(O)OR₃₁, wherein R₃₁ is selected from thegroup consisting of: C₁-C₆ alkyl; C₁-C₆ haloalkyl; C₂-C₆ alkenyl; C₂-C₆alkynyl; C₃-C₁₀ cycloalkyl, wherein the C₃-C₁₀ cycloalkyl is optionallysubstituted with from 1-3 independently selected C₁-C₃ alkyl;—CH₂—C₃-C₁₀ cycloalkyl wherein the C₃-C₁₀ cycloalkyl is optionallysubstituted with from 1-3 independently selected C₁-C₃ alkyl;—CH₂-phenyl, wherein the phenyl is optionally substituted with from 1-2substituents independently selected from C₁-C₃ alkyl, C₁-C₃ haloalkyl,C₁-C₃ alkoxy, C₁-C₃ haloalkoxy, nitro, halo, SO₂Me, cyano, and—OC(O)CH₃; and —CH₂-pyridyl. In certain embodiments, R₃₁ is C₁-C₆ alkyl(e.g., tert-butyl). In other embodiments, R₃ has formula —C(O)R₃₂,wherein R₃₂ is selected from the group consisting of: H; C₁-C₆ alkyl;C₁-C₆ haloalkyl; phenyl, wherein the phenyl is optionally substitutedwith from 1-2 substituents independently selected from C₁-C₃ alkyl;C₁-C₃ haloalkyl; C₁-C₃ alkoxy; C₁-C₃ haloalkoxy; nitro; halo; SO₂Me,cyano; and —OC(O)CH₃; and pyridyl. In certain embodiments, R₃₂ is C₁-C₆alkyl (e.g., —CH₃ or iso-propyl).

In some embodiments, R₄ and R₅ are each independently selected from thegroup consisting of H, C₁-C₆ alkyl, X, and —C₁-C₆ alkylene-X. In certainembodiments, R₄ and R₅ are each independently selected from the groupconsisting of H and C₁-C₆ alkyl. In other embodiments, R₄ and R₅ areeach independently selected from the group consisting of H and —C₁-C₆alkylene-X. In certain embodiments, R₄ and R₅ are H. In otherembodiments, one of R₄ and R₅ is H, and the other is —C₁-C₆ alkylene-X.In certain of these embodiments, the compounds can include one or both(e.g., both) of the following features: (i) alkylene-X is —CH₂—X; and(ii) X is phenyl or heteroaryl including from 5 to 6 ring atoms wherein1, 2, or 3 of the ring atoms are independently selected from the groupconsisting of N, NH, N(C1-C3 alkyl), O, and S; each optionallysubstituted with from 1-3 substituents independently selected from thegroup consisting of halogen, cyano, C₁-C₆ alkyl, hydroxyl, C₁-C₆ alkoxy,and —N(R′)R′.

In other embodiments, R₄ and R₅ taken together form a heterocyclic orheteroaryl ring as defined previously and anywhere herein. In certainembodiments, R₄ and R₅ taken together form a heterocyclic ring, e.g., aring selected from the group consisting of azetidinyl, pyrrolidinyl,pyrazolidinyl, isooxazolidinyl, imidazolidinyl, oxazolidinyl,thiazolidinyl, and isothiazolidinyl. In a particular embodiment, R₄ andR₅ taken together form a pyrrolidinyl ring. In certain embodiments, R₄and R₅ taken together form a heteroaryl ring, e.g., a ring selected fromthe group consisting of imidazolyl, pyrazolyl, oxazolyl, isoxazolyl,thiazolyl, pyridinyl, diazinyl, oxazinyl, and thiazinyl.

In some embodiments, R₁ is H; R₂ is H; R₃ is H; and R₄ and R₅ takentogether form a pyrrolidine ring. In some embodiments, R₁ is H; R₂ is H;R₃ is H; and R₄ and R₅ are H. In some embodiments, R₁ is H or CH₃; R₂ isH or CH₃; R₃ is H; and R₄ and R₅ taken together form a pyrrolidinylring. In some embodiments, R₁ is H or CH₃; R₂ is H or CH₃; R₃ is H; andR₄ and R₅ are H. In some embodiments, R₁ is H or CH₃; R₂ is H or CH₃; R₃is H; and one of R₄ and R₅ is H, and the other is —CH₂—X, wherein X isphenyl or heteroaryl including from 5 to 6 ring atoms wherein 1, 2, or 3of the ring atoms are independently selected from the group consistingof N, NH, N(C1-C3 alkyl), O, and S; each optionally substituted withfrom 1-3 substituents independently selected from the group consistingof halogen, cyano, C₁-C₆ alkyl, hydroxyl, C₁-C₆ alkoxy, and —N(R′)R′. Insome embodiments, R₁ is H or CH₃; R₂ is H or CH₃; R₃ is nitrogenprotecting group (e.g., —C(O)OR₃₁ or —C(O)R₃₂); and R₄ and R₅ takentogether form a pyrrolidinyl ring. In some embodiments, R₁ is H or CH₃;R₂ is H or CH₃; R₃ is nitrogen protecting group (e.g., —C(O)OR₃₁ or—C(O)R₃₂); and R₄ and R₅ are H. In some embodiments, R₁ is H or CH₃; R₂is H or CH₃; R₃ is nitrogen protecting group (e.g., —C(O)OR₃₁ or—C(O)R₃₂); and one of R₄ and R₅ is H, and the other is —CH₂—X, wherein Xis phenyl or heteroaryl including from 5 to 6 ring atoms wherein 1, 2,or 3 of the ring atoms are independently selected from the groupconsisting of N, NH, N(C1-C3 alkyl), O, and S; each optionallysubstituted with from 1-3 substituents independently selected from thegroup consisting of halogen, cyano, C₁-C₆ alkyl, hydroxyl, C₁-C₆ alkoxy,and —N(R′)R′.

In some embodiments (including any of the foregoing embodimentsdescribed above), R₆ is selected from the group consisting of —OH, C₁-C₆alkoxy, —OC(O)—C₁-C₆ alkyl, and —OC(O)phenyl. In certain embodiments(including any of the foregoing embodiments described above), R₆ is —OH.In other embodiments (including any of the foregoing embodimentsdescribed above), R₆ is —NH₂. In some embodiments (including any of theforegoing embodiments described above), R₇ is C₁-C₆ alkyl, e.g., CH₃. Insome embodiments (including any of the foregoing embodiments describedabove), R₆ is —OH or —NH₂, and R₇ is C₁-C₆ alkyl, e.g., CH₃. In someembodiments (including any of the foregoing embodiments describedabove), R_(b) is H.

In some embodiments, the compound is selected from the compoundsdelineated in Table 1 and/or the Examples. In certain embodiments, adisclosed compound includes one having the formula:

The compounds of the present disclosure and formulations thereof mayhave a plurality of chiral centers. Each chiral center may beindependently R, S, or any mixture of R and S. For example, in someembodiments, a chiral center may have an R:S ratio of between about100:0 and about 50:50, between about 100:0 and about 75:25, betweenabout 100:0 and about 85:15, between about 100:0 and about 90:10,between about 100:0 and about 95:5, between about 100:0 and about 98:2,between about 100:0 and about 99:1, between about 0:100 and 50:50,between about 0:100 and about 25:75, between about 0:100 and about15:85, between about 0:100 and about 10:90, between about 0:100 andabout 5:95, between about 0:100 and about 2:98, between about 0:100 andabout 1:99, between about 75:25 and 25:75, and about 50:50. Formulationsof the disclosed compounds comprising a greater ratio of one or moreisomers (i.e., R and/or S) may possess enhanced therapeuticcharacteristic relative to racemic formulations of a disclosed compoundsor mixture of compounds. In some instances, chemical formulas containthe descriptor “-(R)-” or “-(S)-” that is further attached to solidwedge or dashed wedge. This descriptor is intended to show a methinecarbon (CH) that is attached to three other substituents and has eitherthe indicated R or S configuration (see, e.g., Table 1).

Disclosed compounds may provide for efficient cation channel opening atthe NMDA receptor, e.g. may bind or associate with the glutamate site ofthe NMDA receptor to assist in opening the cation channel. The disclosedcompounds may be used to regulate (turn on or turn off) the NMDAreceptor through action as an agonist.

The compounds as described herein may be glycine site NMDA receptorpartial agonists. A partial agonist as used in this context will beunderstood to mean that at a low concentration, the analog acts as anagonist and at a high concentration, the analog acts as an antagonist.Glycine binding is not inhibited by glutamate or by competitiveinhibitors of glutamate, and also does not bind at the same site asglutamate on the NMDA receptor. A second and separate binding site forglycine exists at the NMDA receptor. The ligand-gated ion channel of theNMDA receptor is, thus, under the control of at least these two distinctallosteric sites. Disclosed compounds may be capable of binding orassociating with the glycine binding site of the NMDA receptor. In someembodiments, disclosed compounds may possess a potency that is 10-foldor greater than the activity of existing NMDA receptor glycine sitepartial agonists.

The disclosed compounds may exhibit a high therapeutic index. Thetherapeutic index, as used herein, refers to the ratio of the dose thatproduces a toxicity in 50% of the population (i.e., TD₅₀) to the minimumeffective dose for 50% of the population (i.e., ED₅₀). Thus, thetherapeutic index=(TD₅₀):(ED₅₀). In some embodiments, a disclosedcompound may have a therapeutic index of at least about 10:1, at leastabout 50:1, at least about 100:1, at least about 200:1, at least about500:1, or at least about 1000:1.

Compositions

In other aspects, formulations and compositions comprising the disclosedcompounds and optionally a pharmaceutically acceptable excipient areprovided. In some embodiments, a contemplated formulation comprises aracemic mixture of one or more of the disclosed compounds.

Contemplated formulations may be prepared in any of a variety of formsfor use. By way of example, and not limitation, the compounds may beprepared in a formulation suitable for oral administration, subcutaneousinjection, or other methods for administering an active agent to ananimal known in the pharmaceutical arts.

Amounts of a disclosed compound as described herein in a formulation mayvary according to factors such as the disease state, age, sex, andweight of the individual. Dosage regimens may be adjusted to provide theoptimum therapeutic response. For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for themammalian subjects to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier.

The specification for the dosage unit forms of the invention aredictated by and directly dependent on (a) the unique characteristics ofthe compound selected and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, monostearate salts and gelatin.

The compounds can be administered in a time release formulation, forexample in a composition which includes a slow release polymer. Thecompounds can be prepared with carriers that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Many methods for the preparation of such formulations aregenerally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating thecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In accordance with an alternative aspect of the invention, a compoundmay be formulated with one or more additional compounds that enhance thesolubility of the compound.

Methods

Methods for treating a condition in a patient in need thereof byadministering a therapeutically effective dose of a compound describedherein are provided. In some embodiments, the condition may be a mentalcondition. For example, a mental illness may be treated. In anotheraspect, a nervous system condition may be treated. For example, acondition that affects the central nervous system, the peripheralnervous system, and/or the eye may be treated. In some embodiments,neurodegenerative diseases may be treated.

In some embodiments, the methods include administering a compound totreat patients suffering from autism, anxiety, depression, bipolardisorder, attention deficit disorder, attention deficit hyperactivitydisorder (ADHD), schizophrenia, a psychotic disorder, a psychoticsymptom, social withdrawal, obsessive-compulsive disorder (OCD), phobia,post-traumatic stress syndrome, a behavior disorder, an impulse controldisorder, a substance abuse disorder (e.g., a withdrawal symptom, opiateaddiction, nicotine addiction, and ethanol addition), a sleep disorder,a memory disorder (e.g., a deficit, loss, or reduced ability to make newmemories), a learning disorder, urinary incontinence, multiple systematrophy, progressive supra-nuclear palsy, Friedrich's ataxia, Down'ssyndrome, fragile X syndrome, tuberous sclerosis,olivio-ponto-cerebellar atrophy, cerebral palsy, drug-induced opticneuritis, ischemic retinopathy, diabetic retinopathy, glaucoma,dementia, AIDS dementia, Alzheimer's disease, Huntington's chorea,spasticity, myoclonus, muscle spasm, Tourette's syndrome, epilepsy,cerebral ischemia, stroke, a brain tumor, traumatic brain injury,cardiac arrest, myelopathy, spinal cord injury, peripheral neuropathy,acute neuropathic pain, and chronic neuropathic pain.

In some embodiments, methods of treating a memory disorder associatedwith aging, schizophrenia, special learning disorders, seizures,post-stroke convulsions, brain ischemia, hypoglycemia, cardiac arrest,epilepsy, migraine, AIDS dementia, Huntington's chorea, Parkinson'sdisease, early stage Alzheimer's disease, and Alzheimer's disease arecontemplated.

In certain embodiments, methods for treating schizophrenia are provided.For example, paranoid type schizophrenia, disorganized typeschizophrenia (i.e., hebephrenic schizophrenia), catatonic typeschizophrenia, undifferentiated type schizophrenia, residual typeschizophrenia, post-schizophrenic depression, and simple schizophreniamay be treated using the methods and compositions contemplated herein.Psychotic disorders such as schizoaffective disorders, delusionaldisorders, brief psychotic disorders, shared psychotic disorders, andpsychotic disorders with delusions or hallucinations may also be treatedusing the compositions contemplated herein.

Paranoid schizophrenia may be characterized where delusions or auditoryhallucinations are present, but thought disorder, disorganized behavior,or affective flattening are not. Delusions may be persecutory and/orgrandiose, but in addition to these, other themes such as jealousy,religiosity, or somatization may also be present. Disorganized typeschizophrenia may be characterized where thought disorder and flataffect are present together. Catatonic type schizophrenia may becharacterized where the patient may be almost immobile or exhibitagitated, purposeless movement. Symptoms can include catatonic stuporand waxy flexibility. Undifferentiated type schizophrenia may becharacterized where psychotic symptoms are present but the criteria forparanoid, disorganized, or catatonic types have not been met. Residualtype schizophrenia may be characterized where positive symptoms arepresent at a low intensity only. Post-schizophrenic depression may becharacterized where a depressive episode arises in the aftermath of aschizophrenic illness where some low-level schizophrenic symptoms maystill be present. Simple schizophrenia may be characterized by insidiousand progressive development of prominent negative symptoms with nohistory of psychotic episodes.

In some embodiments, methods are provided for treating psychoticsymptoms that may be present in other mental disorders, including, butnot limited to, bipolar disorder, borderline personality disorder, drugintoxication, and drug-induced psychosis. In another embodiment, methodsfor treating delusions (e.g., “non-bizarre”) that may be present in, forexample, delusional disorder are provided.

Also provided are methods for treating social withdrawal in conditionsincluding, but not limited to, social anxiety disorder, avoidantpersonality disorder, and schizotypal personality disorder.

In some embodiments, methods are provided for treating neuropathic pain.The neuropathic pain may be acute or chronic. In some cases, theneuropathic pain may be associated with a condition such as herpes, HIV,traumatic nerve injury, stroke, post-ischemia, fibromyalgia, reflexsympathetic dystrophy, complex regional pain syndrome, spinal cordinjury, sciatica, phantom limb pain, diabetic neuropathy, and cancerchemotherapeutic-induced neuropathic pain. Methods for enhancing painrelief and for providing analgesia to a patient are also contemplated.

Further contemplated methods include a method of treating autism and/oran autism spectrum disorder in a patient need thereof, comprisingadministering an effective amount of a compound to the patient. In anembodiment, a method for reducing the symptoms of autism in a patient inneed thereof is contemplated, comprising administering an effectiveamount of a disclosed compound to the patient. For example, uponadministration, the compound may decrease the incidence of one or moresymptoms of autism such as eye contact avoidance, failure to socialize,attention deficit, poor mood, hyperactivity, abnormal sound sensitivity,inappropriate speech, disrupted sleep, and perseveration. Such decreasedincidence may be measured relative to the incidence in the untreatedindividual or an untreated individual(s).

Also provided herein is a method of modulating an autism target geneexpression in a cell comprising contacting a cell with an effectiveamount of a compound described herein. The autism gene expression may befor example, selected from ABAT, APOE, CHRNA4, GABRA5, GFAP, GRIN2A,PDYN, and PENK. In another embodiment, a method of modulating synapticplasticity in a patient suffering from a synaptic plasticity relateddisorder is provided, comprising administering to the patient aneffective amount of a compound.

In another embodiment, a method of treating Alzheimer's disease, ore.g., treatment of memory loss that e.g., accompanies early stageAlzheimer's disease, in a patient in need thereof is provided,comprising administering a compound. Also provided herein is a method ofmodulating an Alzheimer's amyloid protein (e.g., beta amyloid peptide,e.g. the isoform Aβ₁₋₄₂), in-vitro or in-vivo (e.g. in a cell)comprising contacting the protein with an effective amount of a compoundis disclosed. For example, in some embodiments, a compound may block theability of such amyloid protein to inhibit long-term potentiation inhippocampal slices as well as apoptotic neuronal cell death. In someembodiments, a disclosed compound may provide neuroprotective propertiesto a Alzheimer's patient in need thereof, for example, may provide atherapeutic effect on later stage Alzheimer's-associated neuronal celldeath.

In a further embodiment, a method of treating depression comprisingadministering a compound described herein is provided. In someembodiments, the treatment may relieve depression or a symptom ofdepression without affecting behavior or motor coordination and withoutinducing or promoting seizure activity. Exemplary depression conditionsthat are expected to be treated according to this aspect of theinvention include, but are not limited to, major depressive disorder,dysthymic disorder, psychotic depression, postpartum depression,premenstrual syndrome, premenstrual dysphoric disorder, seasonalaffective disorder (SAD), bipolar disorder (or manic depressivedisorder), mood disorder, and depressions caused by chronic medicalconditions such as cancer or chronic pain, chemotherapy, chronic stress,and post traumatic stress disorders. In addition, patients sufferingfrom any form of depression often experience anxiety. Various symptomsassociated with anxiety include fear, panic, heart palpitations,shortness of breath, fatigue, nausea, and headaches among others.Anxiety or any of the symptoms thereof may be treated by administering acompound as described herein.

Also provided herein are methods of treating a condition intreatment-resistant patients, e.g., patients suffering from a mental orcentral nervous system condition that does not, and/or has not,responded to adequate courses of at least one, or at least two, othercompounds or therapeutics. For example, provided herein is a method oftreating depression in a treatment resistant patient, comprising a)optionally identifying the patient as treatment resistant and b)administering an effective dose of a compound to said patient.

In some embodiments, a compound described herein may be used for acutecare of a patient. For example, a compound may be administered to apatient to treat a particular episode (e.g., a severe episode) of acondition contemplated herein.

Also contemplated herein are combination therapies comprising a compoundin combination with one or more other active agents. For example, acompound may be combined with one or more antidepressants, such astricyclic antidepressants, MAO-I's, SSRI's, and double and triple uptakeinhibitors and/or anxiolytic drugs. Exemplary drugs that may be used incombination with a compound include Anafranil, Adapin, Aventyl, Elavil,Norpramin, Pamelor, Pertofrane, Sinequan, Surmontil, Tofranil, Vivactil,Parnate, Nardil, Marplan, Celexa, Lexapro, Luvox, Paxil, Prozac, Zoloft,Wellbutrin, Effexor, Remeron, Cymbalta, Desyrel (trazodone), andLudiomill. In another example, a compound may be combined with anantipsychotic medication. Non-limiting examples of antipsychoticsinclude butyrophenones, phenothiazines, thioxanthenes, clozapine,olanzapine, risperidone, quetiapine, ziprasidone, amisulpride,asenapine, paliperidone, iloperidone, zotepine, sertindole, lurasidone,and aripiprazole. It should be understood that combinations of acompound and one or more of the above therapeutics may be used fortreatment of any suitable condition and are not limited to use asantidepressants or antipsychotics.

EXAMPLES

The following examples are provided for illustrative purposes only, andare not intended to limit the scope of the disclosure.

Table 1 below shows some exemplary compounds of the disclosure andprovides physiochemical characteristics of the compounds.

TABLE 1 Molecular Weight Compound Structure (Da) cLogP tPSA Compound X

227 −1.94 96.7 Compound Y

213 −2.36 96.7 Compound Z

281 −1.09 72.9 2S-19

423.5032 0.634639 96.46 2S-20

381.4665 0.193514 90.39 2S-21

341.4027 −0.243061 113.17 2S-24

395.4931 0.610089 90.39 2S-27

395.4931 0.610089 90.39 2S-30

409.5197 1.02666 90.39 2S-8 

383.4824 0.808343 99.18 2S-9 

395.4931 0.789186 99.18 2S-FNL-10

417.4986 1.28851 99.18 2S-FNL-11

317.3828 0.00532027 81.67 2S-FNL-12

359.4195 −0.384737 89.95 2S-FNL-13

387.4727 0.858785 89.95 2S-FNL-14

409.4369 −0.960294 138.1 2S-FNL-15

409.4369 −1.65688 138.1 2S-FNL-16

423.3444 −2.94007 120.59 2S-FNL-17

361.3956 −1.88186 115.73 2S-FNL-18

419.4748 −0.35934 124.96 2S-FNL-19

433.3823 −1.6029 107.45 2S-FNL-2

327.3761 −0.659636 113.17 2S-FNL-20

389.4488 −0.696233 115.73 2S-FNL-21

213.2337 −2.3594 95.66 2S-FNL-22, 2S-16

313.3495 −1.07621 113.17 2S-FNL-23

405.4482 −0.769835 124.96 2S-FNL-24

305.3324 −2.01339 107.45 2S-FNL-25

375.4222 −1.10673 115.73 2S-FNL-26

281.3507 −1.08968 72.88 2S-FNL-27

359.848 −0.648554 78.95 2S-FNL-28

241.2869 −1.52625 95.66 2S-FNL-29

433.5013 0.0511544 124.96 2S-FNL-3

227.2603 −1.94283 95.66 2S-FNL-30

333.3855 −1.1924 107.45 2S-FNL-31

403.4754 −0.285738 115.73 2S-FNL-32

295.3773 −0.673104 72.88 2S-FNL-33

341.4027 −0.243061 113.17 2S-FNL-34

241.2869 −1.52625 95.66 2S-FNL-35

295.3773 −0.673104 72.88 2S-FNL-36

355.4293 0.173514 113.17 2S-FNL-37

255.3134 −1.10968 95.66 2S-FNL-38

309.4039 −0.256529 72.88 2S-FNL-4

297.3501 −1.08936 103.94 2S-FNL-5

426.4689 −0.121843 134.65 2S-FNL-6

326.3913 −0.766518 118.96 2S-FNL-7

262.736 −2.04971 101.45 2S-FNL-8

397.3899 −0.47485 81.67 2S-FNL-9

409.4006 −0.494007 81.67

Example 1—Synthesis of Compound X

Synthesis of (2S,3R)-2-((tert-butoxycarbonyl) amino)-3-hydroxybutanoicacid (A)

To a stirred solution of L-threonine (SM1) (100 g, 0.84 mol) in1,4-dioxane (500 mL) and water (800 mL) was added Na₂CO₃ (178 g, 1.67mol) and stirred at RT for 30 min. The reaction mixture was cooled to 0°C., Boc-anhydride (219.6 g, 1.007 mol) was added drop wise and stirringwas continued for 16 h. After consumption of the starting material (byTLC), the reaction mixture was concentrated under reduced pressure andobtained residue was neutralized using 1N HCl (pH-4). The aqueous layerwas extracted with EtOAc (2×250 mL). The separated organic extracts werewashed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford A (160 g, 87%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 6.30 (d, 1H), 4.07-4.01 (m, 1H), 3.90 (d,1H), 1.99 (s, 1H), 1.42 (s, 9H), 1.09 (d, 3H).

LCMS (m/z): 218.1 [M⁺−1]

Synthesis of (2S,3R)-3-(benzyloxy)-2-((tert-butoxycarbonyl) amino)butanoic acid (B)

To a stirred solution of A (100 g, 0.45 mol) in DMF (600 mL) was added60% NaH (36.5 g, 0.91 mol) portion wise at −20° C. under N₂ atmosphereand stirred for 2 h. To this was added benzyl bromide (66.8 mL, 0.55mol) drop wise and the reaction mixture was stirred at RT for 12 h.After consumption of the starting material (by TLC), the reactionmixture was quenched with ice cold water and washed with diethyl ether(2×250 mL). The separated aqueous layer was acidified using 1N HCl andextracted with EtOAc (2×250 mL). The combined organic layers were driedover anhydrous Na₂SO₄ and concentrated under reduced pressure to affordB (100 g, 71%).

Synthesis of (2S,3R)-benzyl 3-(benzyloxy)-2-((tert-butoxycarbonyl)amino) butanoate (C)

To a stirred solution of B (100 g, 0.32 mol) in DMF (400 mL) was addedK₂CO₃ (111.6 g, 0.81 mol) under N₂ atmosphere and stirred for 30 min. Tothis was added benzyl bromide (47.4 mL, 0.38 mol) drop wise and stirredat RT for 12 h. The reaction mixture was quenched with ice cold waterand extracted with diethyl ether (2×250 mL). The separated the organiclayer was washed with brine, dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The crude material was purified bysilica gel column chromatography eluting with 5% EtOAc/n-hexane toafford C (80 g, 62%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.41-7.25 (m, 10H), 5.09 (s, 2H),4.55-4.50 (m, 1H), 4.34-4.30 (m, 1H), 2.09 (s, 3H), 1.42 (s, 9H), 1.15(d, 3H).

Synthesis of (2S,3R)-benzyl 2-amino-3-(benzyloxy) butanoate (Int-D)

To a stirred solution of C (80 g, 0.20 mol) in methanol (100 mL) wasadded methanolic HCl (70 mL) under N₂ atmosphere and stirred for 12 h.After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure. The crude material waswashed with n-hexane and dried under reduced pressure to afford Int-D(45 g, 75%) as HCl salt.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.35-7.30 (m, 10H), 5.25 (q, 2H),4.58-4.52 (m, 3H), 4.37 (d, 1H), 4.27 (br s, 1H), 4.15-4.10 (m, 1H),1.30 (d, 3H).

LCMS (m/z): 300.2 [M⁺+1]

Synthesis of (S)-methyl pyrrolidine-2-carboxylate (2)

To a stirred solution of L-proline 1 (100 g, 0.87 mol) in methanol (800mL) was added thionyl chloride (76.9 mL, 1.04 mol) slowly drop wise at0° C. The reaction mixture was heated to reflux for 12 h. Afterconsumption of the starting material (by TLC), the reaction wasconcentrated under reduced pressure. The residue was washed withn-hexane to afford 2 (143.9 g, HCl salt).

¹H-NMR: (400 MHz, CDCl₃): δ 3.89 (s, 3H), 3.68-3.62 (m, 2H), 3.59-3.47(m, 2H), 2.49-2.37 (m, 1H), 2.27-2.05 (m, 3H).

LCMS (m/z): 166 [M⁺+1]

Synthesis of (S)-1-tert-butyl 2-methyl pyrrolidine-1,2-dicarboxylate (3)

To a stirred solution of 2 (35 g, 0.22 mol) in CH₂Cl₂ (175 mL) was addedEt₃N (90 mL, 0.65 mol) followed by Boc-anhydride (56.9 mL, 0.26 mol) at0° C. The reaction mixture was stirred at RT for 16 h. After consumptionof the starting material (by TLC), the reaction was diluted with water(100 mL) and extracted with CH₂Cl₂ (2×100 mL). The organic layer waswashed with water, brine, dried over Na₂SO₄ and concentrated. The crudematerial was purified by silica gel column chromatography eluting with30% EtOAc/n-hexane to afford 3 (41 g, 95%).

¹H-NMR: (400 MHz, CDCl₃): δ 4.25-4.21 (m, 1H), 3.75 (s, 3H), 3.57-3.26(m, 2H), 2.29-2.10 (m, 1H), 1.99-1.75 (m, 3H), 1.45 (s, 9H).

LCMS (m/z): 130 [(M⁺+1)-Boc]

Synthesis of 1-tert-butyl 2-methyl 2-((benzyloxy) methyl)pyrrolidine-1,2-dicarboxylate

To a stirred solution of 3 (100 g, 0.43 mol) in THF (800 mL) was addedLiHMDS (873 mL, 0.87 mol) at −78° C. and stirred for 1 h. To thisBOM-chloride (93.2 mL, 0.65 mol) was added drop wise at −78° C. andstirred for 2 h at −20° C. After consumption of the starting material(by TLC), the reaction was quenched with aqueous NH₄Cl solution andextracted with EtOAc. The separated organic layer was washed with water,dried over Na₂SO₄ and concentrated to afford 4 (180 g, crude). Thismaterial was directly taken for the next step without furtherpurification.

LCMS (m/z): 250 [(M⁺+1)-Boc]

Synthesis of 2-((benzyloxy) methyl)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (5)

To a stirred solution of 4 (100 g, 0.28 mol) in methanol (200 mL) wasadded 2N NaOH solution (300 mL) at RT. The reaction mixture was heatedto reflux for 4 h. After consumption of the starting material (by TLC),the solvent from the reaction was evaporated under reduced pressure anddiluted with EtOAc (100 mL). The aqueous layer was acidified usingcitric acid solution and extracted with CH₂Cl₂ (2×250 mL). The separatedorganic layer was washed with water, dried over Na₂SO₄ and concentratedto afford 5 (60 g, 63%).

¹H-NMR: (400 MHz, CDCl₃): δ 7.37-7.32 (m, 5H), 4.61 (s, 2H), 4.05-3.88(m, 2H), 3.65-3.42 (m, 2H), 2.54-2.46 (m, 2H), 1.95 (br s, 2H), 1.57 (s,9H).

LCMS (m/z): 334 [M⁺+1]

Synthesis of1-(tert-butoxycarbonyl)-2-(hydroxymethyl)pyrrolidine-2-carboxylic acid(6)

To a stirred solution of 5 (10 g, 29.81 mmol) in methanol (300 mL) wasadded 50% wet 10% Pd/C (5 g) at RT and stirred for 24 h under H₂atmosphere (balloon pressure). After consumption of the startingmaterial (by TLC), the reaction mixture was filtered through a pad ofcelite and the pad was washed with methanol. Obtained filtrate wasconcentrated under reduced pressure to afford 6 (6 g, 82%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.55 (br m, 1H), 3.99 (d, 1H), 3.88 (d,1H), 7.65-7.60 (m, 1H), 3.51-3.45 (m, 1H), 3.39-3.34 (m, 1H), 2.32-2.14(m, 1H), 1.98-1.69 (m, 3H), 1.39 (s, 9H).

Synthesis of tert-butyl2-(((2S,3R)-1,3-bis(benzyloxy)-1-oxobutan-2-yl)carbamoyl)-2-(hydroxymethyl)pyrrolidine-1-carboxylate (7)

To a stirred solution of 6 (3 g, 12.2 mmol) in CH₂Cl₂ (100 mL) was addedInt-D (5.8 g, 14.6 mmol), EDCI.HCl (2.8 g, 14.6 mmol) followed by HOBt(1.99 g, 14.6 mmol) and DIPEA (4.8 g, 36.7 mmol) at RT and stirred for16 h. After consumption of the starting material (by TLC), the reactionmixture was diluted with water (100 mL) and extracted with CH₂Cl₂ (2×100mL). The separated organic layer was washed with brine, dried overanhydrous Na₇SO₄, filtered and concentrated under reduced pressure.Obtained crude material was purified by silica gel column chromatographyto afford 7 (1.6 g, 25%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.25-8.12 (m, 1H), 7.31-7.27 (m, 10H),5.85 (t, 1H), 5.14 (s, 2H), 4.54-4.49 (m, 2H), 4.31 (dd, 1H), 4.15-4.07(m, 1H), 3.91-3.50 (m, 1H), 3.52-3.37 (m, 1H), 3.31-3.27 (m, 2H),2.35-2.07 (m, 1H), 1.95-1.90 (m, 1H), 1.73-1.52 (m, 2H), 1.39-1.27 (m,9H), 1.19-1.12 (m, 3H).

Mass (ESI): m/z 527.4 [M⁺+1]

Synthesis of tert-butyl2-((2S,3R)-1,3-bis(benzyloxy)-1-oxobutan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(8)

To a stirred solution of 7 (1.4 g, 2.65 mmol) in THF (20 mL) was addedtriphenylphosphine (1.1 g, 3.98 mmol) and DTAD (1.2 g, 3.98 mmol). Thereaction mixture was stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction was concentrated under reducedpressure. The crude material was purified by silica gel columnchromatography to afford 8-F1 (0.6 g) and 8-F2 (0.55 g).

Synthesis of(2S,3R)-2-(5-(tert-butoxycarbonyl)-1-oxo-2,5-diazaspiro[3,4]octan-2-yl)-3-hydroxybutanoicacid (9)

To a stirred solution of 8-F1 and 8-F2 (0.6 g) in methanol (50 mL) wasadded 10% Pd/C (120 mg) at RT and stirred for 6 h under H₂ atmosphere(balloon pressure). After consumption of the starting material (by TLC),the reaction mixture was filtered through a pad of celite and the padwas washed with methanol. The filtrate was concentrated under reducedpressure to give crude, trituration using diethyl ether afforded 9 (0.3g, 82%) as an off-white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.95 (br s, 1H), 4.97 (br s, 1H),4.24-4.20 (m, 1H), 4.14-4.07 (m, 1H), 3.84 (d, 1H), 3.53 (t, 1H),3.41-3.35 (m, 1H), 3.27-3.22 (m, 1H), 2.14-2.08 (m, 2H), 1.84-1.80 (m,2H), 1.42 (s, 9H), 1.24 (d, 3H).

LCMS (m/z): 329.6 [M⁺+1]

Synthesis of tert-butyl2-((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(10)

To a stirred solution of 9 (5 g, 15.2 mmol) in CH₂Cl₂ (100 mL) was addedammonium chloride (2 g, 38.1 mmol), EDCI.HCl (3.5 g, 18.2 mmol) followedby HOBt (5.9 g, 45.7 mmol) and DIPEA (5.9 g, 45.7 mmol) at RT andstirred for 16 h. After consumption of the starting material (by TLC),the reaction mixture was diluted with water (100 mL) and extracted withCH₂Cl₂ (2×100 mL). The separated organic layer was washed with brine,dried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure. The crude material was triturated with Et₂O (50 mL) andn-pentane (50 mL) to afford 10 (2.5 g, 51%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.51 (br s, 1H), 7.19 (br s, 1H), 4.64 (d,1H), 4.07-3.95 (m, 2H), 3.78 (m, 1H), 3.62-3.35 (m, 2H), 3.27-3.25 (m,1H), 2.18-2.05 (m, 2H), 1.86-1.74 (m, 2H), 1.41 (s, 9H), 1.12 (d, 3H).

LCMS (m/z): 328.2 [M⁺+1]

Synthesis of(2S,3R)-3-hydroxy-2-(1-oxo-2,5-diazaspiro[3,4]octan-2-yl)butanamide(Compound X)

To a stirred solution of 10 (2.2 g, 6.70 mmol) in CH₂Cl₂ (25 mL) wasadded TFA (7.6 g, 67 mmol) at 0° C. and stirred at RT for 2 h. Thereaction mixture was concentrated under reduced pressure to affordCompound X (2 g, 87%) as TFA salt.

¹H-NMR: (400 MHz, D₂O): δ 4.33-4.29 (m, 2H), 4.09 (d, 1H), 3.95 (d, 1H),3.57-3.48 (m, 2H), 2.51-2.46 (m, 2H), 2.25-2.19 (m, 2H), 1.31 (d, 3H).

LCMS (m/z): 455 [2M⁺+1]

Example 2—Synthesis of Compound Z

Synthesis of (2S,3R)-2-((tert-butoxycarbonyl) amino)-3-hydroxybutanoicacid (A)

To a stirred solution of (2S,3R)-2-amino-3-hydroxybutanoic acid (SM2)(30 g, 0.25 mol) in THF (150 mL) and water (150 mL) was added NaHCO₃ (65g, 0.75 mol) followed by Boc-anhydride (66 mL, 0.302 mol) at 0° C. Thereaction mixture was stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was extracted withEtOAc (2×150 mL). The aqueous layer was acidified using 2N HCl and thenextracted with 10% MeOH/CH₂Cl₂. The separated organic extracts weredried over anhydrous Na₂SO₄, filtered and concentrated under vacuum toafford A (30 g, 63%).

¹H-NMR: (400 MHz, CDCl₃): δ 5.92-5.70 (m, 2H), 5.55 (d, 1H), 4.42 (br s,1H), 4.29 (d, 1H), 1.47 (s, 9H), 1.25 (d, 3H)

LCMS (m/z): 218 [M⁺−1]

Synthesis of tert-butyl ((2S,3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl) carbamate (B)

To a stirred solution of A (13 g, 59.36 mmol) in DMF (65 mL) was addedEDCI.HCl (12.5 g, 65.2 mmol) followed by HOBt (8.8 g, 65.2 mmol) at 0°C. After stirring for 5 min, DIPEA (30.6 mL, 0.17 mol) followed bypyrrolidine (4.6 g, 65.2 mmol) was added to the reaction mixture andstirring was continued for another 16 h at RT. The reaction mixture waswashed with water and extracted with EtOAc (2×100 mL). The organic layerwas washed with brine, dried over anhydrous Na₂SO₄ and concentratedunder vacuum. The crude was purified by column chromatography to affordB (5 g, 31%).

¹H-NMR: (400 MHz, CDCl₃): δ 5.51 (br s, 1H), 4.32 (d, 1H), 4.15-4.10 (m,1H), 3.77-3.74 (m, 1H), 3.55-3.46 (m, 3H), 1.99-1.94 (m, 2H), 1.91-1.85(m, 2H), 1.47 (s, 9H), 1.26 (t, 1H), 1.29 (d, 3H).

Synthesis of (2R,3S)-3-((tert-butoxycarbonyl)amino)-4-oxo-4-(pyrrolidin-1-yl) butan-2-yl acetate (D)

To a stirred solution of B (4 g, 14.7 mmol) in CH₂Cl₂ (40 mL) was addedEt₃N (5.1 mL, 36.7 mmol) followed by acetic anhydride (1.7 g, 17.6 mmol)and catalytic amount of DMAP at 0° C. The reaction mixture was stirredat RT for 16 h. After consumption of the starting material (by TLC), thereaction mixture was diluted with water and separated the organic layer.Organic layer was washed with water, dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The crude residue obtained waspurified by silica gel column chromatography to give C. To this1,4-dioxane/HCl (20 mL) was added and stirred at RT for 2 h. Thereaction mixture was concentrated under vacuum and the residue waswashed with Et₂O (2×15 mL) to afford D (3.5 g, 97%) as HCl salt.

¹H-NMR: (500 MHz, DMSO-d₆) (Rotamers): δ8.49 (br s, 3H), 8.15 (br s,1H), 5.14-5.10 (m, 1H), 4.26-4.22 (m, 1H), 3.97-3.95 (m, 1H), 3.59 (s,2H), 2.09 (s, 3H), 1.98 (s, 2H), 1.87-1.80 (m, 2H), 1.26 (d, 3H).

LCMS (m/z): 215.1 [M⁺+1]

Synthesis of methyl pyrrolidine-2-carboxylate (1)

To a stirred solution of pyrrolidine-2-carboxylic acid (SM1) (100 g,0.87 mol) in methanol (800 mL) was added thionyl chloride (76.9 mL, 1.04mol) slowly drop wise at 0° C. The reaction mixture was heated to refluxfor 12 h. After consumption of the starting material (by TLC), thereaction was concentrated under vacuum. The residue was washed withn-Hexane and distilled off the solvent to afford 1 (143.9 g, HCl salt).

¹H-NMR: (400 MHz, CDCl₃) (Rotamers): δ 3.89 (s, 3H), 3.68-3.62 (m, 2H),3.59-3.47 (m, 2H), 2.49-2.37 (m, 1H), 2.27-2.05 (m, 3H).

LCMS (m/z): 166 [M⁺+1]

Synthesis of 1-tert-butyl 2-methyl pyrrolidine-1,2-dicarboxylate (2)

To a stirred solution of 1 (35 g, 0.22 mol) in CH₂Cl₂ (175 mL) wereadded Et₃N (90 mL, 0.65 mol) followed by Boc-anhydride (56.9 mL, 0.26mol) at 0° C. The reaction mixture was stirred at RT for 16 h. Afterconsumption of the starting material (by TLC), the reaction was dilutedwith water (100 mL) and extracted with CH₂Cl₂ (2×100 mL). The organiclayer was washed with water, brine, dried over Na₂SO₄ and concentrated.The crude material was purified by silica gel column chromatographyeluting with 30% EtOAc/Hexane to afford 2 (41 g, 95%).

¹H-NMR: (400 MHz, CDCl₃) (Rotamers): δ 4.25-4.21 (m, 1H), 3.75 (s, 3H),3.57-3.26 (m, 2H), 2.29-2.10 (m, 1H), 1.99-1.75 (m, 3H), 1.45 (s, 9H).

LCMS (m/z): 130 [(M⁺+1)-Boc]

Synthesis of 1-tert-butyl 2-methyl 2-((benzyloxy) methyl)pyrrolidine-1,2-dicarboxylate

To a stirred solution of 2 (100 g, 0.43 mol) in THF (800 mL) was addedLiHMDS (873 mL, 0.87 mol) at −78° C. and stirred for 1 h. To thisBOM-chloride (93.2 mL, 0.65 mol) was added drop wise at −78° C. andstirred for 2 h at −20° C. After consumption of the starting material(by TLC), the reaction was quenched with NH₄Cl at 0° C. The separatedorganic layer was washed with water, dried over Na₂SO₄ and concentratedto afford 3 (180 g, crude). This material was directly taken for thenext step without further purification.

LCMS (m/z): 250 [(M⁺+1)-Boc]

Synthesis of 2-((benzyloxy) methyl)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (4)

To a stirred solution of 3 (100 g, 0.28 mol) in methanol (200 mL) wasadded 2N NaOH solution (300 mL) at RT. The reaction mixture was heatedto reflux for 4 h. After consumption of the starting material (by TLC),the solvent from the reaction was evaporated under vacuum and dilutedwith EtOAc (100 mL). The aqueous layer was acidified using citric acidsolution and extracted with CH₂Cl₂ (2×250 mL). The separated organiclayer was washed with water, dried over Na₂SO₄ and concentrated toafford 4 (60 g, 63%).

¹H-NMR: (400 MHz, CDCl₃) (Rotamers): δ 7.37-7.32 (m, 5H), 4.61 (s, 2H),4.05-3.88 (m, 2H), 3.65-3.42 (m, 2H), 2.54-2.46 (m, 2H), 1.95 (br s,2H), 1.57 (s, 9H).

LCMS (m/z): 334 [M⁺−1]

Synthesis of tert-butyl 2-(((2S,3R)-3-acetoxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl) carbamoyl)-2-((benzyloxy) methyl) pyrrolidine-1-carboxylate(5)

To a stirred solution of D (1 g, 2.90 mmol) in DMF (8 mL) was addedEDCI.HCl (0.63 g, 3.28 mmol) followed by HOBt (0.44 g, 3.28 mmol) at 0°C. After being stirred for 5 min, DIPEA (1.3 mL, 7.46 mmol) followed bycompound 4 (0.74 g, 3.58 mmol) was added to the reaction mixture andstirring was continued for another 16 h at RT. The reaction mixture waswashed with water and extracted with EtOAc (2×500 mL). The organic layerwas washed with brine, dried over anhydrous Na₂SO₄ and concentratedunder vacuum. The crude was purified by column chromatography to afford5 (0.6 g, 38%).

LCMS (m/z): 532 [M⁺+1]

Synthesis of tert-butyl 2-(((2S,3R)-3-acetoxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl) carbamoyl)-2-(hydroxymethyl) pyrrolidine-1-carboxylate (6)

To a stirred solution of 5 (4.5 g, 8.40 mmol) in MeOH (40 mL) was addedwet 10% Pd/C (1.5 g) under inert atmosphere and stirred for 4 h under H₂atmosphere (balloon pressure). The reaction mixture was filtered throughcelite pad and concentrated under reduced pressure to afford 6 (3.0 g,81%).

LCMS (m/z): 442.5 [M⁺+1]

Synthesis of tert-butyl 2-((2S,3R)-3-acetoxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate (7)

To a stirred solution of 6 (3 g, 6.70 mmol) in THF (25 mL) was addedtriphenylphosphine (2 g, 7.40 mmol) followed by DTAD (2.5 g, 10.2 mmol).The reaction mixture was stirred at RT for 16 h. After consumption ofthe starting material (by TLC), the reaction was concentrated underreduced pressure. The crude material was purified by silica gel columnchromatography eluting with 10% MeOH/CH₂Cl₂ to afford 7 (1.2 g withTPPO, 43%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 5.25-5.19 (m, 1H), 4.65 (d, 1H), 3.61-3.57(m, 3H), 3.47-3.42 (m, 2H), 3.41-3.25 (m, 4H), 2.05 (s, 4H), 1.95-1.71(m, 7H), 1.42 (s, 10H).

LCMS (m/z): 424.4 [M⁺+1]

Synthesis of(2R,3S)-4-oxo-3-(1-oxo-2,5-diazaspiro[3,4]octan-2-yl)-4-(pyrrolidin-1-yl)butan-2-yl acetate (8)

A stirred solution of 7 (0.4 g, 0.94 mmol) in 1,4-dioxane/HCl (5 mL) wascooled to 0° C. and stirred at RT for 1 h. After consumption of thestarting material (by TLC), the reaction mixture was concentrated underreduced pressure. The crude material was washed with n-pentane followedby EtOAc to afford 8 (0.22 g, 65%).

¹H-NMR: (400 MHz, D₂O): δ 4.62 (d, 1H), 4.41-4.29 (m, 2H), 4.24 (d, 1H),3.89-3.77 (m, 3H), 3.54-3.49 (m, 3H), 2.57-2.52 (m, 1H), 2.49 (s, 3H),2.42-2.00 (m, 8H), 1.30 (d, 3H).

LCMS (m/z): 324.3 [M⁺+1]

UPLC Purity: 99.37%

Synthesis of tert-butyl 2-((2S,3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate (9)

A solution of 7 (0.15 g, 0.41 mmol) in aqueous NH₃ (2 mL) was stirred atRT for 4 h. After consumption of the starting material (by TLC), thereaction diluted with CH₂Cl₂ (75 mL). The separated organic layer wasdried over anhydrous Na₂SO₄ and concentrated under reduced pressure toafford 9 (0.1 g, 76%).

LCMS (m/z): 382 [M¹+1]

Synthesis of 2-((2S,3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-2,5-diazaspiro[3,4]octan-1-one (Compound Z)

To a stirred solution of 9 (0.2 g, 0.63 mmol) in CH₂Cl₂ (2 mL) was addedTFA (0.3 mL) at 0° C. and stirred at RT for 1 h. The reaction mixturewas concentrated under vacuum and the residue was diluted with water andextracted with CH₂Cl₂ (2×25 mL). The separated organic layer was driedover anhydrous Na₂SO₄, filtered and concentrated under vacuum to affordCompound Z (0.2 g, 80%) as TFA salt.

¹H-NMR: (400 MHz, D₂O): δ 4.64 (t, 1H), 4.25-4.21 (m, 1H), 4.09 (d, 1H),3.99-3.87 (m, 1H), 3.70 (t, 2H), 3.55-3.47 (m, 5H), 2.52-2.34 (m, 2H),2.25-2.22 (m, 2H), 2.08-1.98 (m, 5H), 1.25 (t, 3H).

LCMS (m/z): 282.4 [M⁺+1]

Synthesis of (2S,3R)-2-((tert-butoxycarbonyl) amino)-3-hydroxybutanoicacid (2S-A)

To a stirring solution of L-threonine (50 g, 420 mol) in THF/water (500mL/500 mL) were added NaHCO₃ (111 g, 1.05 mol) and stirred at RT for 30min. The reaction mixture was cooled to 0° C. and Boc-anhydride (137 mL,630 mmol) was added drop wise and the stirring was continued at RT for16 h. After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure and obtained residue wasdiluted with water (100 mL) and acidified by using 1N HCl (pH-3). Theaqueous layer was extracted with EtOAc (2×250 mL). The combined organiclayer was washed with brine (1×200 mL), dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure to afford compound 2S-A(80 g, 87%) as thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.5 (br s, 1H), 6.30 (d, J=8.5 Hz, 1H),4.50 (br s, 1H), 4.05-4.02 (m, 1H), 3.88-3.86 (m, 1H), 1.39 (s, 9H),1.08 (d, J=6.0 Hz, 3H); LCMS m/z: 218.1 [M⁺−1]

Synthesis of (2S,3R)-3-(benzyloxy)-2-((tert-butoxycarbonyl) amino)butanoic acid (2S-B)

To a stirring solution of 2S-A (40 g, 182 mmol) in DMF (400 mL) wasadded 60% NaH (18.2 g, 758 mmol) portion wise at −20° C. under N₂atmosphere and stirred for 2 h. To this added benzyl bromide (66.8 mL,0.55 mol) drop wise and the reaction mixture was stirred at RT for 3 h.After consumption of the starting material (by TLC), the reactionmixture was quenched with ice cold water and washed with diethyl ether(2×250 mL). The separated aqueous layer was acidified using citric acidsolution (100 mL) and extracted with EtOAc (2×250 mL). The combinedorganic layers were dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to afford compound 2S-B (45 g, 80%) as thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.64 (br s, 1H), 7.34-7.25 (m, 5H), 6.46(d, J=8.5 Hz, 1H), 4.53 (d, J=11.5 Hz, 1H), 4.39 (d, J=12.0 Hz, 1H),4.00-3.98 (m, 2H), 1.39 (s, 9H), 1.15 (d, J=6.0 Hz, 3H);

Synthesis of (2S,3R)-benzyl 3-(benzyloxy)-2-((tert-butoxycarbonyl)amino) butanoate (2S-C)

To a stirring solution of compound 2S-B (45 g, 146 mmol) in DMF (400 mL)was added K₂CO₃ (40 g, 292 mmol) under N₂ atmosphere and stirred for 30min. To this benzyl bromide (21 mL, 175 mmol) was added drop wise at 0°C. and stirred at RT for 16 h. The reaction mixture was quenched withice cold water and extracted with diethyl ether (2×250 mL). Theseparated organic layer was washed with brine, dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The crude material waspurified by silica gel column chromatography eluting with 20%EtOAc/n-hexane to afford compound 2S-C (48 g, 82%) as thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.37-7.18 (m, 10H), 6.81 (d, J=9.0 Hz,1H), 5.08 (s, 2H), 4.49 (d, J=12.0 Hz, 1H), 4.32 (d, J=12.0 Hz, 1H),4.25-4.22 (m, 1H), 4.01-3.98 (m, 1H), 1.38 (s, 9H), 1.15 (d, J=6.0 Hz,3H)

Mass (ESI): m/z 399.4[M′+1];

Synthesis of (2S,3R)-benzyl 2-amino-3-(benzyloxy) butanoate (2S-D)

To a stirring solution of compound 2S-C (48 g, 120 mmol) in diethylether(50 mL) was added diethylether saturated with HCl (350 mL) at 0° C. andstirred at RT for 10 h.

After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure. The crude material wastriturated with diethyl ether/n-pentane (50 mL/50 mL) and dried underreduced pressure to afford compound 2S-D (28 g, 77%) as semisolid (HClsalt).

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.59 (s, 2H), 7.50-7.25 (m, 10H), 5.23 (d,J=12.5 Hz, 1H), 5.16 (d, J=12.5 Hz, 1H), 4.54 (d, J=12.0 Hz, 1H), 4.36(d, J=12.0 Hz, 1H), 4.12-4.09 (m, 1H), 4.09-3.99 (m, 1H), 1.29 (d, J=6.5Hz, 3H)

Mass (ESI): m/z 299.4[M⁺+1];

Synthesis of methyl pyrrolidine-2-carboxylate (2S-E)

To a stirring solution of L-proline (50 g, 434 mmol) in methanol wasadded thionyl chloride (37.5 ml, 521 mmol) at 0° C. and heated to 70° C.for 16 h. The reaction mixture was brought to RT and concentrated undervacuum to afford compound 2S-E as (70 g, 99%) as thick syrup(hydrochloride salt).

¹H-NMR: (500 MHz, DMSO-d₆): δ 4.15-4.13 (m, 1H), 3.65 (s, 3H), 3.35-3.30(m, 2H), 2.23-2.15 (m, 1H), 1.86-1.78 (m, 3H), 1.41 (s, 9H);

LCMS m/z: 129 [M⁺+1]

Synthesis of 1-tert-butyl 2-methyl pyrrolidine-1,2-dicarboxylate (2S-F)

To a stirring solution of compound 2S-E (70 g, 422 mmol) in CH₂Cl₂ (700mL) were added Et₃N (183 mL, 1.26 mol) at 0° C. and stirred for 10 min.After added Boc-anhydride (184 mL, 845 mmol) at 0° C. and the reactionmixture was stirred at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction was diluted with water (200 mL) andextracted with CH₂Cl₂ (2×200 mL). The combined organic layer was washedwith citric acid (1×150 mL), brine (1×200 mL). The organic layer wasdried over Na₂SO₄ and concentrated under reduced pressure to affordcrude compound which was purified by column chromatography by eluting50% EtOAC/n-hexane to obtain compound 2S-F (80 g, 83%) as thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 4.15-4.13 (m, 1H), 3.65 (s, 3H), 3.35-3.30(m, 2H), 2.23-2.15 (m, 1H), 1.86-1.78 (m, 3H), 1.41 (s, 9H);

LCMS m/z: 229 [(M⁺+1)-Boc].

Synthesis of 1-tert-butyl 2-methyl 2-((benzyloxy) methyl)pyrrolidine-1,2-dicarboxylate (2S-G)

To a stirring solution of compound 2S-F (25 g, 109 mmol) in THF (250 mL)was added LiHMDS (240 mL, 240 mmol) at −20° C. and stirred for 2 h. Tothis BOM-chloride (23 mL, 163 mmol) was added drop wise at −30° C. andstirred for 2 h. After consumption of the starting material (by TLC),the reaction was quenched with aqueous NH₄Cl solution (100 mL) andextracted with EtOAc (2×200 mL). The combined organic layer was washedwith water (2×150 mL) followed by brine solution (2×100 mL). The organiclayer was dried over Na₂SO₄ and concentrated to obtain crude compoundwhich was purified by column chromatography by eluting 10%EtOAc/n-hexane to afford compound 2S-G (30 g, 79%) as thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.36-7.22 (m, 5H), 4.59-4.48 (m, 2H),4.02-3.88 (m, 1H), 3.63 (s, 3H), 3.49-3.35 (m, 2H), 3.34-3.30 (m, 1H),2.31-2.23 (m, 1H), 2.04-1.89 (m, 2H), 1.82-1.78 (m, 1H); LCMS m/z: 349.4[(M′+1)-Boc]

Synthesis of 2-((benzyloxy) methyl)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (2S-H)

To a stirring solution of compound 2S-G (30 g, 86 mmol) in methanol (70mL) was added NaOH solution (6.88 g in 70 mL H₂O) at RT. The reactionmixture was heated to 70° C. for 16 h. After consumption of the startingmaterial (by TLC), the solvent from the reaction was evaporated underreduced pressure and diluted with EtOAc (2×200 mL). The separatedaqueous layer was acidified using citric acid solution (pH-3) andextracted with EtOAc (2×250 mL). The combined organic layer was driedover Na₂SO₄ and concentrated to afford crude was triturated withn-hexane to obtain compound 2S-H (25 g, 86.8%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.35 (br s, 1H), 7.37-7.29 (m, 5H),4.56-4.48 (m, 2H), 4.06-4.00 (m, 1H), 3.92-3.89 (m, 1H), 3.66-3.45 (m,1H), 3.37-3.28 (m, 1H), 2.31-2.20 (m, 1H), 2.05-1.97 (m, 1H), 1.87-1.75(m, 2H), 1.38 (s, 9H); LCMS m/z: 335.3 [M⁺+1]

Synthesis of 1-(tert-butoxycarbonyl)-2-(hydroxymethyl)pyrrolidine-2-carboxylic acid (2S-I)

To a stirring solution of compound 2S-H (25 g, 74 mmol) in methanol (150mL) was added 50% wet 10% Pd/C (7 g) at RT and stirred for 10 h under H₂atmosphere. After consumption of the starting material (by TLC), thereaction mixture was filtered through a pad of celite and the pad waswashed with methanol (100 mL). Obtained filtrate was concentrated underreduced pressure to afford compound 2S-I (15 g, 82.8%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 4.66 (br s, 1H), 3.96-3.83 (m, 1H),3.63-3.59 (m, 1H), 3.49-3.41 (m, 1H), 3.34-3.25 (m, 1H), 2.30-2.17 (m,1H), 1.95-1.72 (m, 3H), 1.38 (s, 9H).

Mass (ESI): m/z 245 [M⁺+1]

Synthesis of tert-butyl 2-(((2S,3R)-1,3-bis (benzyloxy)-1-oxobutan-2-yl)carbamoyl)-2-(hydroxymethyl) pyrrolidine-1-carboxylate (2S-J)

To a stirring solution of compound 2S-I (18 g, 73.4 mmol) in CH₂Cl₂ (180mL) were added DIPEA (40 mL, 220 mmol), 2S-D (21.9 g, 73.4 mmol), HATU(41.8 g, 110 mmol) at RT and stirred for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was diluted with water(50 mL) and extracted with CH₂Cl₂ (2×100 mL). The combined organic layerwas washed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. Obtained crude material waspurified by silica gel column chromatography eluting with 30%EtOAc/n-hexane to afford compound 2S-J (20 g, 52%) as pale yellow thicksyrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.25-8.12 (m, 1H), 7.31-7.27 (m, 10H),5.85 (t, J=4.8 Hz, 1H), 5.14 (s, 2H), 4.54-4.49 (m, 2H), 4.31-4.20 (m,1H), 4.15-4.07 (m, 1H), 3.91-3.50 (m, 1H), 3.52-3.37 (m, 1H), 3.31-3.27(m, 2H), 2.35-2.07 (m, 1H), 1.95-1.90 (m, 1H), 1.73-1.52 (m, 2H), 1.39(s, 9H), 1.19 (d, J=6.4 Hz, 3H);

Mass (ESI): m/z 527.4 [M⁺+1]

Synthesis of tert-butyl 2-((2S,3R)-1,3-bis(benzyloxy)-1-oxobutan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-K)

To a stirring solution of triphenylphosphine (24.7 g, 94 mmol) in THF(100 mL) was added DIAD (15.3 g, 75 mmol) at RT and stirred for 30 min.To this added compound 2S-J (20 g, 37.9 mmol) in (10 mL) THF slowly andreaction mixture was stirred at RT for 2 h. After consumption of thestarting material (by TLC), the reaction was concentrated under reducedpressure. The crude material was purified by silica gel columnchromatography eluting 25% EtOAc/n-hexane to afford compound 2S-K (17 g,88%) as pale yellow thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.33-7.26 (m, 5H), 7.23-7.18 (m, 5H), 5.10(s, 2H), 4.80-4.73 (m, 2H), 4.60 (s, 2H), 4.31 (s, 2H), 4.05-4.00 (m,2H), 1.80-1.68 (m, 4H), 1.39 (s, 9H), 1.18 (d, J=6.0 Hz, 3H);

Mass (ESI): m/z 509.4 [M⁺+1]

Synthesis of(2S,3R)-2-(5-(tert-butoxycarbonyl)-1-oxo-2,5-diazaspiro[3,4]octan-2-yl)-3-hydroxybutanoicacid (2S-L)

To a stirring solution of compound 2S-K (7 g, 13.7 mmol) in methanol(100 mL) was added 10% Pd/C (4 g) at RT and stirred for 6 h under H₂atmosphere. After consumption of the starting material (by TLC), thereaction mixture was filtered through a pad of celite and the pad waswashed with methanol (50 mL). Obtained filtrate was concentrated underreduced pressure to obtained crude, which was triturated with n-pentane(50 mL) to afford compound 2S-L (4 g, 88%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.80 (br s, 1H), 4.78-4.73 (m, 1H),4.21-4.19 (m, 1H), 4.09 (s, 2H), 3.55-3.46 (m, 2H), 2.09-2.05 (m, 2H),1.80 (d, J=7.0 Hz, 1H), 1.38 (s, 9H), 1.35-1.28 (m, 2H), 1.17 (d, J=6.5Hz, 3H)

LCMS m/z: 329.6 [M⁺+1]

Synthesis of 2-((tert-butoxycarbonyl) amino) acetic acid (2S-M)

To a stirring solution of glycine (15 g, 200 mmol) in 1,4-dioxane/water(150 mL/75 mL) were added Na₂CO₃ (53 g, 500 mmol). After addedBoc-anhydride (109 mL, 500 mmol) slowly at 0° C. The reaction mixturewas stirred at RT for 12 h. After consumption of the starting material(by TLC), the reaction mixture was concentrated under reduced pressure.The crude residue was acidified (pH-4) by using citric acid solution andaqueous layer was extracted with EtOAc (2×150 mL). The combined organiclayer was washed with brine solution (2×100 mL). The organic layer wasdried over anhydrous Na₂SO₄, filtered and concentrated under vacuum toafford compound 2S-M (30 g, 85.7%) as white solid. This material wasdirectly used for the next step without further purification.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.41 (br s, 1H), 7.04 (t, J=5.5 Hz, 1H),3.57 (d, J=5.5 Hz, 2H), 1.37 (s, 9H);

Synthesis of tert-butyl (2-amino-2-oxoethyl) carbamate (2S-N)

To a stirring solution of 2S-M (10 g, 57.14 mmol) in CH₂Cl₂ (100 mL)were added HOBt (15.43 g, 114 mmol), EDCI.HCl (21.8 g, 114 mmol)followed by NH₄Cl (4.54 g, 85.71 mmol) and DIPEA (30.7 mL, 171 mmol) at0° C. The reaction mixture was stirred at RT for 16 h. After consumptionof the starting material (by TLC), the reaction mixture was washed withwater (2×100 mL). Organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to give crude; which was purified bysilica gel column chromatography eluting with 2% MeOH/CH₂Cl₂. Aftercompound was triturated with ether (25 mL) and the precipitated solidwas filtered to afford 2S-N (2 g, 20%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.52 (br s, 1H), 7.17 (br s, 1H), 3.46 (d,J=6.5 Hz, 2H), 1.38 (s, 9H);

Synthesis of (E)-tert-butyl(2-(((dimethylamino)methylene)amino)-2-oxoethyl) carbamate (2S-O)

To a stirring solution of 2S-N (7 g, 40.22 mmol) in THF (70 mL) wasadded DMF.DMA (10.7 mL, 80.44 mmol) at RT and heated to 80° C. for 2 h.After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure to afford 2S-O (9 g,crude) as brown syrup. This crude material was directly taken for thenext step without further purification.

¹H-NMR: (500 MHz, DMSO-d₆): δ 6.72 (br s, 1H), 4.35 (s, 1H), 3.64 (d,J=5.5 Hz, 2H), 3.09 (s, 1H), 1.42 (s, 9H);

Mass (ESI): m/z 230.2 [M⁺+1];

Synthesis of tert-butyl ((1,2,4-oxadiazol-5-yl) methyl) carbamate (2S-P)

To a stirring solution of 2S-O (9 g (crude), 39.30 mmol) in ethanol (80mL) was added hydroxylamine hydrochloride (5.45 g, 78.60 mmol) under N₂atmosphere. The reaction mixture was heated to 90° C. and stirred for 2h. After consumption of the starting material (by TLC) evaporatedsolvent under reduced pressure and crude residue was diluted with water(75 mL). The aqueous layer was extracted by DCM (3×100 mL). The combinedorganic layer was washed by brine solution (1×100 mL). The organic layerwas dried over anhydrous Na₂SO₄ and solvent was concentrated underreduced pressure to give crude; which was purified by silica gel columnchromatography eluting with 25% EtOAc/hexane to afford 2S-P (4 g, 51%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.90 (s, 1H), 7.64 (s, 1H), 4.44 (s, 2H),1.39 (s, 9H);

LCMS m/z: 198.4 [M⁻−1]

Synthesis of (1,2,4-oxadiazol-5-yl) methanamine (2S-O)

To a stirring solution of 2S-P (1.1 g, 5.52 mmol) in DCM (30 mL) wasadded trifluoroacetic acid (2.1 mL, 27.63 mmol) at 0° C. for 30 min. Thereaction mixture was stirred at RT for 4 h. After consumption of thestarting material (by TLC), the reaction mixture was concentrated undervacuum. The crude residue was triturated with ether (20 mL) to afford2S-Q (850 mg, 72.6%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.13 (s, 1H), 8.90 (br s, 2H), 4.56 (s,2H);

LCMS (ESI): 100.4 [M¹+1]

Synthesis of methyl (tert-butoxycarbonyl) glycinate (2S-R)

To a stirring solution of glycine methyl ester hydrochloride (50 g, 400mmol) in 1,4 dioxane/water (300 mL/200 mL) were added Na₂CO₃ (84.8 g,800 mmol) and stirred at RT for 10 min. The reaction mixture was cooledto 0° C. and Boc-anhydride (104 mL, 480 mmol) was added drop wise andthe stirring was continued at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was concentrated underreduced pressure and obtained residue was diluted with water (100 mL)and extracted with EtOAc (2×250 mL). The combined organic layer waswashed with brine (1×200 mL) and organic layer was dried over anhydrousNa₇SO₄, filtered and concentrated under reduced pressure to afford 2S-R(64 g, 84%) as thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.19 (t, J=5.5 Hz, 1H), 3.67 (d, J=6.0 Hz,2H), 3.62 (s, 3H), 1.38 (s, 9H);

LCMS m/z: 190.2 [M⁺+1]

Synthesis of tert-butyl (2-hydrazinyl-2-oxoethyl) carbamate (2S-S)

A solution of 2S-R (20 g, 105 mmol) in EtOH (100 mL) was added hydrazinehydrate (15.8 g, 315 mmol) at RT and after stirred at 100° C. for 6 h.After consumption of the starting material (by TLC), ethanol wasevaporated under reduced pressure. Obtained crude material wastriturated with n-pentane/diethyl ether (20 mL/20 mL) to afford 2S-S aswhite solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.91 (s, 1H), 6.88 (t, J=5.5 Hz, 1H), 4.16(s, 2H), 3.47 (d, J=6.0 Hz, 2H), 1.37 (s, 9H);

LCMS m/z: 190.2 [M⁺+1]

Synthesis of tert-butyl ((1,3,4-oxadiazol-2-yl) methyl) carbamate (2S-T)

A solution of 2S-S (14 g, 74 mmol) in triethyl orthoformate (140 mL) wasadded p-TSA (catalytic, 140 mg) at RT and after stirred at 80° C. for 4h. After consumption of starting material (by TLC), triethylorthoformate was evaporated under reduced pressure. The crude residuewas purified by column chromatography eluting 20% EtOAc/hexane to afford2S-T (6.1 g, 41.5%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 10.74 (s, 1H), 7.45 (s, 1H), 4.03 (s, 2H),1.47 (s, 9H);

LCMS m/z: 200.2 [M⁺+1]

Synthesis of (1,3,4-oxadiazol-2-yl) methanamine (2S-U)

To a stirring solution of 2S-T (5 g, 25 mmol) in EtOAc (10 mL) was addedEtOAc saturated with HCl (60 mL) at 0° C. and stirred at RT for 16 h.After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure. The crude material wastriturated with diethylether/n-pentane (25 mL/25 mL) and dried underreduced pressure to afford 2S-U (3 g, 88.7%) as an off-white solid (HClsalt).

¹H-NMR: (500 MHz, DMSO-d₆): δ 9.55 (br s, 2H), 7.99 (s, 1H), 3.90 (s,2H);

LCMS m/z: 100 [M⁺+1]

Synthesis of (azidomethyl) benzene (2S-V)

To a stirring solution of benzyl bromide (30 g, 175 mmol) in dimethylformamide (300 mL) was added sodium azide (45.6 g, 701 mmol) at RT underinert atmosphere. The resultant reaction mixture was stirred at 70° C.for 16 h. After completion of reaction monitored (by TLC), the reactionmixture was allowed to RT; the volatiles were diluted with water (300mL) and ether (200 mL). The separated organic layer was washed by (3×200mL) of chilled water. The separated organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toafford compound 2S-V (18 g, crude) as an off-white solid.

¹H-NMR: (400 MHz, CDCl₃): δ 7.40-7.29 (m, 5H), 4.32 (s, 2H).

Synthesis of ethyl 1-benzyl-5-methyl-1H-1,2,3-triazole-4-carboxylate(2S-W2)

To a stirring solution of ethyl but-2-ynoate (8.0 g, 71.3 mmol) intoluene (80 mL) was added 2S-V (12.0 g, 107 mmol) at RT under inertatmosphere. The resultant reaction mixture was heated to 100° C. andstirred for 16 h. The reaction mixture was allowed to RT; the volatileswere evaporated under reduced pressure to which, crude residue waspurified by column chromatography by eluting 40% EtOAc/hexane to afford2S-W1 and 2S-W2 (8.2 g, 47.1%) (separable by column chromatography)

¹H-NMR: (400 MHz, CDCl₃): δ 7.36-7.31 (m, 3H), 7.16 (t, J=6.0 Hz, 2H),5.53 (s, 2H), 4.43 (q, J=7.2 Hz, 2H), 2.45 (s, 3H), 1.41 (t, J=7.2 Hz,3H);

Mass m/z: 246.3 [M⁺+1]

Synthesis of 1-benzyl-5-methyl-1H-1,2,3-triazole-4-carboxylic acid(2S-X2)

To a stirring solution of compound 2S-W2 (8.2 g, 33.4 mmol) in THF/H₂O(82 mL/82 mL, 1:1) was added LiOH.H₂O (4.2 g, 0.4 mmol) at RT andstirred for 16 h. After completion of reaction (by TLC), the volatileswere evaporated under reduced pressure. The residue was acidified withaqueous 2N HCl and the precipitated solid was filtered and washed withwater (25 mL), dried under reduced pressure to afford compound 2S-X2(7.0 g, 96.6%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 13.01 (br s, 1H), 7.40-7.32 (m, 5H), 5.63(s, 2H), 2.45 (s, 3H);

Mass m/z: 218.3 [M⁺+1];

Synthesis of pyrimidin-2-ylmethanamine (2S-Y)

To a stirring solution of 2-cyanopyrimidine (2.0 g, 19.0 mmol) inmethanol (50 mL) were added 10% Pd/C (300 mg), 12 N HCl (1.5 mL) underN₂ atmosphere. The reaction mixture was stirred under H₂ atmosphere(balloon pressure) at RT for 3 h. After consumption of the startingmaterial (by TLC), the reaction mixture was filtered through a pad ofcelite and the pad was washed with methanol. Obtained filtrate wasconcentrated under reduced pressure to afford crude compound which wastriturated with diethyl ether to obtained compound 2S-Y (1.2 g, 44%) aswhite solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.87 (d, J=5.0 Hz, 2H), 8.69 (br s, 2H),7.52 (t, J=5.0 Hz, 1H), 4.24 (s, 2H);

Mass (ESI): 110.3 [M⁺+1]

Synthesis of (S)-2-((tert-butoxycarbonyl) amino)-3-hydroxypropanoic acid(2S-Z)

To a stirring solution of L-serine (76 g, 723 mmol) in 1,4 dioxane/H₂O(350 mL/300 mL) were added NaOH (61 g, 1.51 mol), Boc-anhydride (190 mL,868 mmol) at 0° C. The reaction mixture was stirred at RT for 16 h.After consumption of the starting material (by TLC), the reactionmixture was acidified with 2N HCl (pH-4) and extracted with EtOAc (5×500mL). The combined organic extracts were dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to afford 2S-Z (100 g, 67.5%) asyellow syrup.

¹H-NMR: (400 MHz, CDCl₃): δ 6.54 (br s, 1H), 5.77 (br s, 1H), 4.35-4.04(m, 1H), 3.87-3.84 (m, 2H), 1.45 (s, 9H).

Synthesis of (S)-3-(benzyloxy)-2-((tert-butoxycarbonyl) amino) propanoicacid (2S-AA)

To a stirring solution of 2S-Z (50 g, 245 mmol) in DMF (650 mL) wasadded NaH (60%) (23 g, 563 mmol) at −15° C. and stirred for 2 h. Benzylbromide (32.8 mL, 269 mmol) was slowly added. The reaction mixturetemperature was warmed to RT and stirred for 12 h. After consumption ofthe starting material (by TLC), the reaction mixture was poured intochilled water (200 mL) and extracted with diethylether (2×250 mL). Theaqueous layer was acidified with citric acid (pH-4) and extracted withEtOAc (2×500 mL). The combined organic layers were washed with water(3×250 mL). The organic extracts were dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure to afford 2S-AA (54 g,75%) as brown syrup.

¹H-NMR: (400 MHz, CDCl₃): δ 7.32-7.26 (m, 5H), 5.43 (d, J=7.6 Hz, 1H),4.70-4.46 (m, 1H), 4.45 (s, 2H), 4.13-3.91 (m, 1H), 3.73-3.70 (m, 1H),1.44 (s, 9H).

Synthesis of (S)-benzyl 3-(benzyloxy)-2-((tert-butoxycarbonyl) amino)propanoate (2S-AB)

To a stirring solution of 2S-AA (36 g, 122 mmol) in DMF (250 mL) wasadded Na₂CO₃ (20 g, 183 mmol) at 0° C. and added benzyl bromide (18 mL,146 mmol) slowly. The reaction mixture temperature was warmed to RT andstirred for 12 h. After consumption of the starting material (by TLC),the reaction mixture was poured into chilled water (200 mL) andextracted with diethylether (2×250 mL). The combined organic layers werewashed with water (3×250 mL). The organic extracts were dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toafford 2S-AB (42 g, 91%) as brown syrup was used directly for next stepwithout any purification.

Synthesis of (S)-benzyl 2-amino-3-(benzyloxy) propanoate hydrochloride(2S-AC)

To a stirring solution of 2S-AB (10 g, 25.9 mmol) in ether saturatedwith HCl (50 mL) was added at 0° C. and stirred at RT for 12 h. Theobtained precipitate was filtered and triturated with diethylether(2×100 mL). The filtered compound was dried under vacuum to afford 2S-AC(5 g, 60%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.66 (s, 2H), 7.38-7.27 (m, 10H),5.29-5.22 (m, 2H), 4.57-4.44 (m, 3H), 3.91-3.81 (m, 2H)

Synthesis of tert-butyl 2-(((S)-1,3-bis (benzyloxy)-1-oxopropan-2-yl)carbamoyl)-2-(hydroxymethyl) pyrrolidine-1-carboxylate (2S-AD)

To a stirring solution of compound 2S-I (5 g, 20.4 mmol) in CH₂Cl₂ (50mL) were added DIPEA (10.7 mL, 61.2 mmol), 2S-AC (5.8 g, 20.4 mmol),HATU (11.6 g, 30.6 mmol) at 0° C. and stirred to RT for 12 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with water (100 mL) and extracted with CH₂Cl₂ (2×100 mL). Thecombined organic layer was washed with citric acid (1×100 mL) followedby brine solution (1×100 mL). The organic layer was dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure. Obtained crudematerial was purified by silica gel column chromatography eluting with50% EtOAc/n-hexane to afford compound 2S-AD (8 g, 76.5%) as yellow thicksyrup.

¹H-NMR: (400 MHz, CD₃OD): δ 7.33-7.24 (m, 10H), 5.23-5.11 (m, 2H),4.72-4.66 (m, 2H), 4.50-4.44 (m, 1H), 4.18-3.91 (m, 2H), 3.75-3.70 (m,2H), 3.65-3.40 (m, 2H), 2.34-2.03 (m, 2H), 1.81-1.78 (m, 2H), 1.41 (s,9H);

Mass (ESI): m/z 512.6 [M⁺+1]

Synthesis of tert-butyl 2-((S)-1,3-bis(benzyloxy)-1-oxopropan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-AE)

To a stirring solution of triphenylphosphine (640 mg, 2.44 mmol) in THF(5 mL) was added DIAD (392 mg, 1.94 mmol) at RT and stirred for 15 min,then compound 2S-AD (500 mg, 0.97 mmol) in (5 mL) THF was slowly addedand reaction mixture was stirred at RT for 2 h. After consumption of thestarting material (by TLC), the reaction was concentrated under reducedpressure. The crude material was purified by column chromatography byeluting 2% MeOH/DCM to afford compound 2S-AE (450 mg, 93%) as yellowliquid.

¹H-NMR: (400 MHz, CD₃OD): δ 7.34-7.27 (m, 10H), 5.25-5.14 (m, 2H),4.78-4.73 (m, 1H), 4.70-4.42 (m, 2H), 4.04-3.98 (m, 1H), 3.93-3.78 (m,1H), 3.89-3.78 (m, 1H), 3.76-3.68 (m, 1H), 3.45-3.35 (m, 2H), 2.20-2.09(m, 2H), 1.90-1.78 (m, 2H), 1.46 (s, 9H)

LCMS (ESI): m/z 495.5 [M⁺+1]

Synthesis of 1-(tert-butoxycarbonyl)-2-(hydroxymethyl)pyrrolidine-2-carboxylic acid (2S-AF)

To a stirring solution of compound 2S-AE (500 mg, 1.01 mmol) in methanol(25 mL) was added 50% wet 10% Pd/C (250 mg) at RT and stirred for 24 hunder H₂ atmosphere. After consumption of the starting material (byTLC), the reaction mixture was filtered through a pad of celite and thepad was washed with methanol (20 mL). Obtained filtrate was concentratedunder reduced pressure to afford compound 2S-AF (400 mg, crude) as whitesolid.

¹H-NMR: (400 MHz, CD₃OD): δ 4.92-4.87 (m, 1H), 4.28-4.07 (m, 3H),3.63-3.60 (m, 1H), 3.55-3.40 (m, 2H), 2.30-2.25 (m, 2H), 1.95-1.87 (m,2H), 1.47 (s, 9H);

LCMS: 315.3 [M+1]

Synthesis of (S)-methyl 2-amino-3-hydroxypropanoate (2S-AG)

To a stirring solution of L-serine (40 g, 0.38 mol) in methanol (300 mL)was added SOCl₂ (33.6 mL, 0.45 mol) drop wise at 0° C. and stirred for 1h. The resulting reaction mixture was refluxed for 24 h. Afterconsumption of the starting material (by TLC), the reaction mixture waswarmed to RT and concentrated under vacuum and decanted with n-hexane(2×200 mL) to afford compound 2S-AG (59.18 g, crude).

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.62 (s, 3H), 4.08 (d, J=3.2 Hz, 1H), 3.83(d, J=3.6 Hz, 2H), 3.78 (s, 3H);

LCMS, m/z: 120.2 [M⁺−1]

Synthesis of (S)-methyl2-(((benzyloxy)carbonyl)amino)-3-hydroxypropanoate (2S-AH)

To a stirring solution of compound 2S-AG (40 g, 0.33 mol) in 1,4-dioxane(300 mL) and water (100 mL) was added Na₂CO₃ (71.18 g, 0.67 mol) andstirred at RT for 30 min. The reaction mixture was cooled to 0° C.,benzyl chloroformate (68.5 g, 0.40 mol) was added drop wise and thestirring was continued at RT for 8 h. After consumption of the startingmaterial (by TLC), the reaction mixture was diluted with EtOAc (200 mL).The aqueous layer was extracted with EtOAc (2×200 mL). The separatedorganic extracts were washed with brine, dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure. The crude material waspurified by silica gel column chromatography eluting with 20%EtOAc/n-hexane to afford compound 2S-AH (53 g, 62%);

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.49 (d, J=8 Hz, 1H), 7.37-7.29 (m, 5H),5.04 (s, 2H), 4.93 (t, J=6 Hz, 1H), 4.18-4.13 (m, 1H), 3.78 (s, 3H),3.67-3.56 (m, 2H)

Synthesis of (S)-methyl2-(((benzyloxy)carbonyl)amino)-3-((tert-butyldiphenylsilyl)oxy)propanoate(2S-AI)

To a stirring solution of compound 2S-AH (20 g, 79.20 mmol) in DCM (700mL) was added imidazole (16 g, 237.6 mmol) at 0° C. followed by TBDPS(25.9 g, 95.04 mmol) under N₂ atmosphere and stirred at RT for 8 h.After consumption of the starting material (by TLC), the reactionmixture was diluted with water (100 mL) and the aqueous layer wasextracted with DCM (2×200 mL). The separated organic layer was washedwith brine, dried over anhydrous Na₂SO₄ and concentrated under reducedpressure. The crude material was purified by silica gel columnchromatography eluting with 20% EtOAc/n-hexane to afford compound 2S-AI(25 g, 64%).

¹H-NMR: (500 MHz, CDCl₃): δ 7.23-7.68 (m, 1H), 7.58 (d, J=7 Hz, 3H),7.44-7.37 (m, 9H), 7.34 (d, J=7.5 Hz, 2H), 5.65 (d, J=9 Hz, 1H), 5.12(d, J=2, 2H), 4.45 (d, J=9 Hz, 1H), 4.10-4.07 (m, 1H), 3.91-3.88 (m,1H), 3.74 (s, 3H), 1.04 (s, 9H);

LCMS (m/z): 492.1 [M⁺−1]

Synthesis of (S)-methyl2-amino-3-((tert-butyldiphenylsilyl)oxy)propanoate (2S-AJ)

To a stirring solution of compound 2S-AI (25 g, 51.12 mmol) in ethanol(250 mL) was added 50% wet 10% Pd/C (15 g) at RT and stirred for 8 hunder H₂ atmosphere (balloon pressure). After consumption of thestarting material (by TLC), the reaction mixture was filtered through apad of celite and the pad was washed with ethanol. Obtained filtrate wasconcentrated under reduced pressure to afford compound 2S-AJ (18 g, 97%)as yellow liquid.

¹H-NMR: (400 MHz, CDCl₃): δ 7.66-7.61 (m, 4H), 7.43-7.36 (m, 6H),4.00-3.97 (m, 2H), 3.74 (s, 3H), 3.64 (t, J=4 Hz, 1H), 2.65 (s, 2H),1.04 (s, 9H);

LCMS m/z: 358 [M⁺−1]

Synthesis of ethyl pyrrolidine-2-carboxylate hydrochloride (2S-AK)

To a stirring solution of L-proline (110 g, 956.5 mmol) in ethanol wasadded thionyl chloride (141 ml, 1911.3 mmol) and refluxed for 16 h. Thereaction mixture was brought to RT and concentrated under vacuum toafford compound 2S-AK as the hydrochloride salt (170 g, 99%).

¹H-NMR: (400 MHz, CDCl₃): δ 4.15-4.10 (m, 2H), 3.68-3.62 (m, 2H),3.59-3.47 (m, 2H), 2.49-2.37 (m, 1H), 2.27-2.05 (m, 3H), 1.18 (t, J=3.6Hz, 3H);

LCMS, m/z: 143 [M⁺+1]

Synthesis of 1-tert-butyl 2-ethyl pyrrolidine-1,2-dicarboxylate (2S-AL)

To a stirring solution of compound 2S-AK (70 g, 0.391 mol) in CH₂Cl₂(700 mL) were added Et₃N (170.7 mL, 1.22 mol) followed by Boc-anhydride(133 g, 0.61 mol) at 0° C. The reaction mixture was stirred at RT for 12h. After consumption of the starting material (by TLC), the reaction wasdiluted with water (100 mL) and extracted with CH₂Cl₂ (2×200 mL). Theorganic layer was washed with water (1×150 mL), brine (1×200 mL), driedover Na₂SO₄ and concentrated under reduced pressure to afford compound2S-AL (90 g, 90%) as thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 4.15-4.10 (m, 2H), 4.09-4.02 (m, 1H),3.36-3.29 (m, 2H), 2.25-2.13 (m, 1H), 1.87-1.76 (m, 3H), 1.40 (s, 9H),1.18 (t, J=3.6 Hz, 3H);

LCMS, m/z: 144 [(M⁺+1)-Boc];

HPLC: 96.11%

Synthesis of 1-tert-butyl 2-ethyl 2-(1-hydroxyethyl)pyrrolidine-1,2-dicarboxylate (2S-AM)

To a stirring solution of compound 2S-AL (5 g, 20.5 mmol) in THF (50 mL)was added LiHMDS (20.3 mL, 20.5 mmol) at −20° C. and stirred for 1 h. Tothis acetaldehyde (1.2 mL, 20.5 mmol) was added dropwise at −20° C. andstirred for 1 h at −20° C. After consumption of the starting material(by TLC), the reaction was quenched with aqueous NH₄Cl solution andextracted with EtOAc (1×50 mL). The separated organic layer was driedover Na₂SO₄ and concentrated to afford crude compound was purified bycolumn chromatography eluting 10% EtOAc/hexane to afford compound 2S-AM(1.8 g, 30%) as pale yellow syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 5.10 (d, J=8.5 Hz, 1H), 4.54-4.36 (m, 2H),4.05-3.99 (m, 2H), 3.60-3.49 (m, 1H), 1.97-1.74 (m, 4H), 1.40 (s, 9H),1.18, 1.15 (dd, J=7.5 Hz, 6.5 Hz, 3H), 0.96 (d, J=9.5 Hz, 3H);

LCMS, m/z: 188 [(M⁺⁺1)-Boc]

Synthesis of 1-(tert-butoxycarbonyl)-2-(1-hydroxyethyl)pyrrolidine-2-carboxylic acid (2S-AN)

To a stirring solution of compound 2S-AM (10 g, 34.8 mmol) in methanol(30 mL) were added NaOH (2.7 g, 69.6 mmol), H₂O/THF (30 mL/30 mL)) at 0°C. The reaction mixture was heated to 80° C. for 5 h. After consumptionof the starting material (by TLC), the solvent was evaporated underreduced pressure. The aqueous layer was acidified using citric acidsolution and extracted with EtOAc (2×100 mL). The separated organiclayer was washed with water (1×50 mL), dried over Na₂SO₄ andconcentrated to afford compound 2S-AN (4.8 g, 53.3%) as brown stickysolid.

¹H-NMR: (500 MHz, DMSO-do): δ 4.60-4.54 (m, 1H), 3.98 (d, J=10.0 Hz,1H), 3.90-3.77 (m, 2H), 3.44-3.34 (m, 1H), 2.01-1.68 (m, 4H), 1.40 (s,9H), 1.26 (d, J=10.0 Hz, 3H);

LCMS, m/z: 258 (M⁺−1);

HPLC (purity): 91.7%

Synthesis of tert-butyl2-(((2S,3R)-1,3-bis(benzyloxy)-1-oxobutan-2-yl)carbamoyl)-2-(1-hydroxyethyl)pyrrolidine-1-carboxylate(2S-AO)

To a stirring solution of compound 2S-AN (2.0 g, 7.72 mmol) in CH₂Cl₂(50 mL) were added DIPEA (4.2 mL, 22.4 mmol), EDCI.HCl (2.2 g, 11.5mmol) followed by HOBt (1.5 g, 11.5 mmol), compound D (2.8 g, 8.35 mmol)at 0° C. and stirred for 12 h. After consumption of the startingmaterial (by TLC), the reaction mixture was diluted with water (30 mL)and extracted with CH₂Cl₂ (2×50 mL). The combined organic layer waswashed with brine (2×50 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. Obtained crude material waspurified by silica gel column chromatography eluting 25% EtOAc/n-hexaneto afford compound 2S-AO (1.5 g, 36%) as colorless liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.47 (t, J=8.8 Hz, 1H), 7.31-7.19 (m,10H), 5.73-5.58 (m, 1H), 5.18 (s, 2H), 4.64 (s, 2H), 4.60-4.49 (m, 1H),4.29 (d, J=12.0 Hz, 1H), 4.15-4.12 (m, 1H), 3.59-3.59 (m, 1H), 3.24-3.13(m, 1H), 1.71-1.60 (m, 2H), 1.43-1.38 (m, 2H), 1.35 (s, 9H), 1.18 (d,J=6.0 Hz, 3H), 1.04 (d, 0.1=6.4 Hz, 3H);

Mass (ESI): m/z 540 [M⁻−1]

Synthesis of tert-butyl2-((2S,3R)-1,3-bis(benzyloxy)-1-oxobutan-2-yl)-1-methyl-3-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-AP)

To a stirring solution of triphenylphosphine (1.45 g, 5.53 mmol) in THF(30 mL) was added DIAD (1.12 g, 5.53 mmol) at RT and stirred for 30 min.To this added compound 2S-AO (1.5 g, 2.77 mmol) in (10 mL) THF slowlyand reaction mixture was stirred at RT for 2 h. After consumption of thestarting material (by TLC), the reaction was concentrated under reducedpressure. The crude material was purified by silica gel columnchromatography eluting 20% EtOAc/hexane to afford compound 2S-AP (800mg, 57%) as pale yellow syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.33-7.18 (m, 10H), 5.07 (s, 2H), 4.61 (s,2H), 4.38-4.31 (m, 1H), 3.77-3.75 (m, 1H), 3.28-3.24 (m, 1H), 2.67-2.66(m, 1H), 2.22-2.12 (m, 1H), 1.98-1.92 (m, 3H), 1.72-1.60 (m, 1H), 1.40(s, 9H), 1.18 (d, J=5.6 Hz, 3H), 1.13 (d, J=6.4 Hz, 3H)

Mass (ESI): m/z 523 [M⁺+1]

Synthesis of(2S,3R)-2-(5-(tert-butoxycarbonyl)-1-methyl-3-oxo-2,5-diazaspiro[3,4]octan-2-yl)-3-hydroxybutanoicacid (2S-AO)

To a stirring solution of compound 2S-AP (900 mg) in methanol (30 mL)was added 10% Pd/C (300 mg) at RT and stirred for 16 h under H₂atmosphere (balloon pressure). After consumption of the startingmaterial (by TLC), the reaction mixture was filtered through a pad ofcelite and washed with methanol (10 mL). Obtained filtrate wasconcentrated under reduced pressure to afford compound 2S-AQ (480 mg,82%) as yellow thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ12.80 (br s, 1H), 5.11-4.96 (m, 1H),4.83-4.04 (m, 3H), 3.40-3.35 (m, 1H), 2.11 (s, 3H), 2.10-2.03 (m, 2H),1.46 (s, 9H), 1.43-1.39 (m, 6H).

LCMS: 342 [M⁻−1]

Synthesis of ethyl 5-oxopyrrolidine-2-carboxylate (2S-AR)

To a stirring solution of 5-oxopyrrolidine-2-carboxylic acid (10 g, 77.4mmol) in ethanol (100 mL) was added thionyl chloride (6.7 mL, 92.9 mmol)at 0° C. The reaction mixture was stirred at RT for 16 h. Afterconsumption of the starting material (by TLC), the solvents from thereaction mixture were removed under vacuum. The residue was diluted with

EtOAc (50 mL) and stirred over K₂CO₃. The organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. Obtained crudematerial was purified by silica gel column chromatography to affordcompound 2S-AR (9 g, 74%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.98 (br s, 1H), 4.16 (t, 3H), 2.37-2.30(m, 1H), 2.15 (q, 2H), 2.03-1.97 (m, 1H), 1.22 (t, 3H);

LCMS, m/z: 157.9 [M⁺+1]

Synthesis of 1-tert-butyl 2-ethyl 5-oxopyrrolidine-1,2-dicarboxylate(2S-AS)

To a stirring solution of compound 2S-AR (9 g, 57.3 mmol) in CH₂Cl₂ (90mL) was added DMAP (7.0 g, 57.3 mmol) followed by Et₃N (15.9 mL, 114.6mmol) and Hoc-anhydride (36.7 mL, 171.9 mmol) at 0° C. The reactionmixture was stirred at RT for 16 h. The reaction mixture was dilutedwith CH₂Cl₂ (50 mL) and washed with aqueous 1N HCl solution followed bybrine. The separated organic layer was dried over anhydrous Na₂SO₄ andconcentrated under vacuum. Obtained crude material was purified bycolumn chromatography eluting with 50% EtOAc/Hexane to afford compound2S-AS (12 g, 82%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 4.61 (dd, 1H), 4.19 (q, 2H), 2.46-2.40 (m,2H), 2.37-2.25 (m, 1H), 1.91-1.85 (m, 1H), 1.42 (s, 9H), 1.22 (t, 3H).

Synthesis of ethyl 2-((tert-butoxycarbonyl) amino)-5-oxohexanoate(2S-AT)

To a stirring solution of compound 2S-AS (12 g, 46.6 mmol) in THF (120mL) under inert atmosphere was added MeMgBr (3M in ether) (20.2 mL, 60.6mmol) at 0° C. and stirred for 2 h. After consumption of the startingmaterial (by TLC), the reaction mixture was quenched with aqueous NH₄Clsolution and the aqueous layer was extracted with EtOAc (2×200 mL). Thecombined organic extracts were dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The crude residue obtained waspurified by silica gel column chromatography eluting with 20%EtOAc/Hexane to afford compound 2S-AT (10 g, 79%).

¹H-NMR: (400 MHz, CDCl₃): δ 5.14 (br s, 1H), 4.23 (q, 2H), 2.62-2.47 (m,2H), 2.17 (s, 4H), 1.91-1.82 (m, 1H), 1.45 (s, 10H), 1.26 (t, 3H).

Synthesis of ethyl 5-methylpyrrolidine-2-carboxylate (2S-AU & 2S-AV)

To a stirring solution of compound 2S-AT (10 g, 36.7 mmol) in CH₂Cl₂(100 mL) was added TFA (14.89 mL, 194.6 mmol) at 0° C. After beingstirred for 2 h at RT, the reaction mixture was concentrated underreduced pressure to get compound 2S-AU. Obtained material was dissolvedin ethanol (100 mL) and 10% Pd/C (50% wet, 3 g) under N₂ atmosphere. Thereaction mixture was stirred under H₂ atmosphere (balloon pressure) for16 h. The reaction mixture was filtered through a pad of celite andfiltrate was concentrated under reduced pressure to afford compound2S-AV (15 g, crude). This material was directly taken for the next stepwithout further purification.

LCMS, m/z: 158.1 [M⁺+1]

Synthesis of 1-tert-butyl 2-ethyl 5-methylpyrrolidine-1,2-dicarboxylate(2S-AW)

To a stirring solution of compound 2S-AV (30 g, 191 mmol) in CH₂Cl₂ (150mL) was added DMAP (23.3 g, 191 mmol) followed by Et₃N (79.8 mL, 573mmol) and Hoc-anhydride (104 mL, 477 mmol) at 0° C. The reaction mixturewas stirred at RT for 16 h. The reaction mixture was diluted with CH₂Cl₂(50 mL) and washed with water (2×150 mL) followed by brine. Theseparated organic layer was dried over anhydrous Na₂SO₄ and concentratedunder vacuum. Obtained crude material was purified by columnchromatography eluting with 6% EtOAc/hexane to afford compound 2S-AW (30g, 61.22%) as pale yellow liquid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 4.13-3.86 (m, 4H), 2.15 (d, J=3.5 Hz, 1H),1.99-1.82 (m, 2H), 1.52 (t, J=4.5 Hz, 1H), 1.38 (s, 9H), 1.24 (t, J=5.5Hz, 3H), 1.16 (d, J=6.5 Hz, 3H). LCMS, m/z: 258 [(M⁺⁺1)

Synthesis of 1-tert-butyl 2-ethyl 2-((benzyloxy)methyl)-5-methylpyrrolidine-1,2-dicarboxylate (2S-AX)

To a stirring solution of compound 2S-AW (8.0 g, 31.12 mmol) in THF (70mL) was added LiHMDS (59 mL, 41.72 mmol) at −78° C. and stirred for 2 h.To this BOM-chloride (6.56 mL, 41.72 mmol) was added dropwise andstirred for 2 h at −30° C. After consumption of the starting material(by TLC), the reaction was quenched with aqueous NH₄Cl solution (20 mL)and extracted with DCM (30 mL). The separated organic layer was driedover Na₂SO₄ and concentrated to afford crude material was purified bycolumn chromatography eluting with 10% EtOAc/Hexane to afford compound2S-AX (11 g, 94.2%) as pale yellow liquid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.33-7.25 (m, 5H), 4.38 (d, J=10.5 Hz,2H), 4.08-3.98 (m, 1H), 3.88 (d, J=9.5 Hz, 2H), 2.20-2.08 (m, 2H), 1.38(s, 9H), 1.37-1.29 (m, 4H), 1.19 (t, J=7.5 Hz, 3H), 1.14-1.10 (m, 3H);

LCMS, m/z: 378 (M⁺+1)

Synthesis of 2-((benzyloxy)methyl)-1-(tert-butoxycarbonyl)-5-methylpyrrolidine-2-carboxylic acid(2S-AY)

To a stirring solution of compound 2S-AX (11 g, 29.17 mmol) in CH₃OH/THF(22 mL/20 mL) were added 2N NaOH solution (33 mL) at RT. The reactionmixture was heated to 65° C. for 8 h. After consumption of the startingmaterial (by TLC), the solvent from the reaction was evaporated underreduced pressure and diluted with EtOAc (50 mL). The aqueous layer wasacidified using citric acid solution and extracted with CH₂Cl₂ (2×100mL). The separated organic layer was washed with water (1×50 mL), driedover Na₂SO₄ and concentrated to afford compound 2S-AY (8 g, 80%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.58 (s, 1H), 7.34-7.28 (m, 5H),4.54-4.47 (m, 2H), 4.05-3.87 (m, 2H), 3.70-3.62 (m, 1H), 2.28-2.08 (m,3H), 1.46-1.37 (m, 1H), 1.28 (s, 9H);

LCMS, m/z: 350 [M⁺+1].

Synthesis of1-(tert-butoxycarbonyl)-2-(hydroxymethyl)-5-methylpyrrolidine-2-carboxylicacid (2S-AZ)

To a stirring solution of compound 2S-AY (8 g, 1.45 mmol) in methanol(40 mL) was added 10% Pd/C (4 g) under N₂ atmosphere. The reactionmixture was stirred under H₂ atmosphere (balloon pressure) at RT for 16h. After consumption of the starting material (by TLC), the reactionmixture was filtered through a pad of celite and the pad was washed withmethanol. Obtained filtrate was concentrated under reduced pressure toafford crude compound which was triturated with n-pentane to obtainedcompound 2S-AZ (4.5 g, 75.2%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.37 (br s, 1H), 4.61 (br s, 1H),3.95-3.85 (m, 3H), 2.18-2.06 (m, 3H), 1.44-1.41 (m, 1H), 1.38 (s, 9H),1.09 (d, J=6.0 Hz, 3H);

LCMS (ESI): m/z 260 [M⁺+1]

Synthesis of tert-butyl 2-(((2S,3R)-1,3-bis (benzyloxy)-1-oxobutan-2-yl)carbamoyl)-2-(hydroxymethyl)-5-methylpyrrolidine-1-carboxylate (2S-BA)

To a stirring solution of compound 2S-AZ (3 g, 11.58 mmol) in DCM (30mL) were added N,N-diisopropylethylamine (6 mL, 34.7 mmol), Int D (5 g,13.8 mmol), followed by EDCI (2.7 g, 13.8 mmol), HOBT (1.9 g, 13.8 mmol)at 0° C. and stirred at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was diluted with water (20 mL).The separated organic layer was washed with saturated NaHCO₃ solution(1×50 mL), 2N HCl solution (30 mL) followed by brine solution (1×40 mL).The separated organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to afford crude compound which waspurified by column chromatography to obtained compound 2S-BA (2 g,32.5%) as pale yellow liquid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.30-7.17 (m, 10H), 5.16-5.10 (m, 2H),4.50 (t, J=5.2 Hz, 2H), 4.28 (t, J=12.0 Hz, 1H), 4.13-4.07 (m, 1H), 3.95(s, 2H), 2.10-1.85 (m, 4H), 1.40-1.35 (m, 1H), 1.30 (s, 9H), 1.18, 1.16(dd, J=6.4 Hz, 6H); LCMS (ESI): m/z 440.3 [M⁺+1]

Synthesis of tert-butyl2-((2S,3R)-1,3-bis(benzyloxy)-1-oxobutan-2-yl)-6-methyl-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-BB)

To a stirring solution of compound 2S-BA (1.0 g, 1.85 mmol) in THF (10mL) was added triphenylphosphine (0.935 g, 4.62 mmol) and DIAD (0.75 g,3.70 mmol). The reaction mixture was stirred at RT for 8 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasconcentrated under reduced pressure. The crude material was purified bysilica gel column chromatography eluting 20% EtOAc/hexane to affordcompound 2S-BB (0.8 g, 51%) as yellow liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.30-7.17 (m, 10H), 5.17 (s, 2H), 4.79,4.76 (dd, J=6.0 Hz, 2H), 4.73 (s, 2H), 4.31-4.18 (m, 2H), 3.84 (t, J=6.8Hz, 2H), 2.12 (t, J=6.8 Hz, 1H), 1.98-1.91 (m, 2H), 1.39 (s, 9H), 1.32,1.25 (dd, J=6.0 Hz, 6.4 Hz, 3H), 1.18, 1.09 (dd, J=6.0 Hz, 6.4 Hz, 3H);

LCMS (ESI): m/z 523.3 [M⁺+1]

Synthesis of(2S,3R)-2-(5-(tert-butoxycarbonyl)-6-methyl-1-oxo-2,5-diazaspiro[3,4]octan-2-yl)-3-hydroxybutanoicacid (2S-BC)

To a stirring solution of compound 2S-BB (1 g, 1.91 mmol) in methanol(20 mL) was added 10% Pd/C (400 mg) under N₂ atmosphere. The reactionmixture was stirred under H₂ atmosphere (balloon pressure) at RT for 16h. After consumption of the starting material (by TLC), the reactionmixture was filtered through a pad of celite and the pad was washed withmethanol. Obtained filtrate was concentrated under reduced pressure toafford compound 2S-BC (0.80 g, crude) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.75 (br s, 1H), 4.80-4.73 (m, 3H),4.20-4.05 (m, 2H), 3.40, 3.36 (dd, J=6.8 Hz, 1H), 2.21-2.13 (m, 1H),2.06-1.99 (m, 2H), 1.53 (t, J=6.0 Hz, 1H), 1.40 (s, 9H), 1.11, 1.10 (dd,J=4.8 Hz, 5.2 Hz, 6H); LCMS (ESI): m/z 343.3 [M⁺+1]

Synthesis of 1-tert-butyl 2-ethyl2-(1-hydroxyethyl)-5-methylpyrrolidine-1,2-dicarboxylate (2S-BD)

To a stirring solution of compound 2S-AW (20 g, 77.8 mmol) in THF (200mL) was added LiHMDS (84 mL, 155 mmol) dropwise at −20° C. and stirredfor 30 min. To this acetaldehyde (8.77 mL, 155 mmol) was added drop wiseand stirred at RT for 3 h. After consumption of the starting material(by TLC), the reaction was quenched with aqueous NH₄Cl solution (100 mL)and extracted with DCM (2×150 mL). The combined organic layer was washedwith brine solution (1×150 mL). The separated organic layer was driedover Na₂SO₄ and concentrated to afford crude material was purified bycolumn chromatography eluting with 30% EtOAc/Hexane to afford compound2S-BD (16 g, 23.1%) as colorless syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.33-7.25 (m, 5H), 4.38 (d, J=10.5 Hz,2H), 4.08-3.98 (m, 1H), 3.88 (d, J=9.5 Hz, 2H), 2.20-2.08 (m, 2H), 1.38(s, 9H), 1.37-1.29 (m, 4H), 1.19 (t, J=7.5 Hz, 3H), 1.14-1.10 (m, 3H);

LCMS m/z: 378 (M⁺+1)

Synthesis of1-(tert-butoxycarbonyl)-2-(1-hydroxyethyl)-5-methylpyrrolidine-2-carboxylicacid (2S-BE)

To a stirring solution of compound 2S-BD (15 g, 49 mmol) in EtOH/THF (10mL/20 mL) were added NaOH (3.98 g, 99 mmol) in water (10 mL) at RT. Thereaction mixture was heated to 90° C. for 4 h. After consumption of thestarting material (by TLC), the solvent from the reaction was evaporatedunder reduced pressure and acidified by using citric acid (pH˜4). Theaqueous layer was extracted with DCM (2×200 mL) and the combined organiclayer was washed with brine solution (1×150 mL). The separated organiclayer was dried over Na₂SO₄ and concentrated to obtained crude compound,which was purified by column chromatography eluting 40% EtOAc/n-hexaneto afford compound 2S-BE (8.2 g, 60.7%) as brown syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.15 (br s, 2H), 4.54-4.50 (m, 1H),4.03-4.02 (m, 1H), 2.17-1.77 (m, 3H), 1.41 (s, 9H), 1.39-1.09 (m, 3H),0.99-0.94 (m, 3H);

LCMS m/z: 272.4 [M⁻−1]

Synthesis of tert-butyl2-(2S,3R)-1,3-bis(benzyloxy)-1-oxobutan-2-yl)carbamoyl)-2-(1-hydroxyethyl)-5-methylpyrrolidine-1-carboxylate(2S-BF)

To a stirring solution of compound 2S-BE (8 g, 29.3 mmol) in DCM (100mL) were added N,N-diisopropylethylamine (15.12 mL, 87 mmol), 2S-D(12.13 g, 40.6 mmol) followed by HATU (16.5 g, 43.5 mmol) at 0° C. andstirred at RT for 12 h. After consumption of the starting material (byTLC), the reaction mixture was diluted with water (100 mL). Theseparated organic layer was washed with citric acid solution (1×75 mL)followed by brine solution (1×100 mL). The organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to afford crudecompound which was purified by column chromatography by eluting with 40%EtOAc/n-hexane to obtain compound 2S-BF (11 g, 68.4%) as pale yellowliquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.31-7.19 (m, 10H), 5.14-5.06 (m, 2H),4.59-4.48 (m, 3H), 4.31-4.26 (m, 1H), 4.05-4.00 (m, 2H), 1.98-1.89 (m,2H), 1.41 (s, 9H), 1.39-1.35 (m, 3H), 1.28-1.17 (m, 6H), 1.16-1.00 (m,3H);

LCMS (ESI): 555.6 [M⁺+1]

Synthesis of tert-butyl2-((2S,3R)-1,3-bis(benzyloxy)-1-oxobutan-2-yl)-1,6-dimethyl-3-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-BG)

To a stirring solution of triphenylphosphine (3.5 g, 13.5 mmol) in THF(10 mL) was added DIAD (2.72 g, 13.5 mmol) as portion-wise and stirredfor 20 min at RT. To this added compound 2S-BF (3 g, 5.4 mmol) in THF(10 mL) slowly at RT and stirred for 3 h. After consumption of thestarting material (by LCMS), the reaction mixture was concentrated underreduced pressure. The crude material was purified by silica gel columnchromatography eluting 20% EtOAc/hexane to afford compound 2S-BG (2.5 g,86.5%) as yellow liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ7.39-7.18 (m, 10H), 5.19-5.10 (m, 2H),4.78-4.49 (m, 3H), 4.34-4.25 (m, 2H), 3.85-3.76 (m, 1H), 2.10-1.69 (m,4H), 1.40 (s, 9H), 1.35-1.26 (m, 3H), 1.18-1.12 (m, 6H);

Synthesis of(2S,3R)-2-(5-(tert-butoxycarbonyl)-1,6-dimethyl-3-oxo-2,5-diazaspiro[3,4]-octan-2-yl)-3-hydroxybutanoicacid (2S-BH)

To a stirring solution of compound 2S-BG (2 g, 3.72 mmol) in methanol(20 mL) was added 10% dry Pd/C (200 mg) under N₂ atmosphere. Thereaction mixture was stirred under H₂ atmosphere at RT for 12 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasfiltered through a pad of celite and the pad was washed with methanol(10 mL). Obtained filtrate was concentrated under reduced pressure toafford compound 2S-BH (1.8 g, 60.4%) as yellow solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.72 (br s, 1H), 5.11-4.97 (m, 1H),4.30-4.15 (m, 2H), 3.91-3.76 (m, 2H), 2.18-1.90 (m, 3H), 1.40 (s, 9H),1.37-1.29 (m, 1H), 1.26-1.22 (m, 3H), 1.21-1.10 (m, 6H);

LCMS (ESI): 357.5 [M⁺+1]

Synthesis of tert-butyl ((2S,3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl) carbamate (2S-BI)

To a stirring solution of compound 2S-A (13 g, 59.36 mmol) in DMF (65mL) was added EDCI.HCl (12.5 g, 65.2 mmol) followed by HOBt (8.8 g, 65.2mmol) at 0° C. After being stirred for 5 min, DIPEA (30.6 mL, 0.17 mol)followed by pyrrolidine (4.6 g, 65.2 mmol) was added to the reactionmixture and stirring was continued for another 16 h at RT. The reactionmixture was washed with water and extracted with EtOAc (2×100 mL). Theorganic layer was washed with brine, dried over anhydrous Na₂SO₄ andconcentrated under vacuum. The crude was purified by columnchromatography to afford compound 2S-BI (5 g, 31%).

¹H-NMR: (400 MHz, CDCl₃): δ5.51 (br s, 1H), 4.32 (d, 1H), 4.15-4.10 (m,1H), 3.77-3.74 (m, 1H), 3.55-3.46 (m, 3H), 1.99-1.94 (m, 2H), 1.91-1.85(m, 2H), 1.47 (s, 9H), 1.26 (t, 1H), 1.29 (d, 3H).

Synthesis of (2R,3S)-3-((tert-butoxycarbonyl)amino)-4-oxo-4-(pyrrolidin-1-yl) butan-2-yl acetate (2S-BJ and 2S-BK)

To a stirring solution of compound 2S-BI (4 g, 14.7 mmol) in CH₂Cl₂ (40mL) was added Et₃N (5.1 mL, 36.7 mmol) followed by acetic anhydride (1.7g, 17.6 mmol) and catalytic amount of DMAP at 0° C. The reaction mixturewas stirred at RT for 16 h. After consumption of the starting material(by TLC), the reaction mixture was diluted with water and separated theorganic layer. Organic layer was washed with water, dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The crude residueobtained was purified by silica gel column chromatography to givecompound 2S-BJ. To this 1,4-dioxane/HCl (20 mL) was added and stirred atRT for 2 h. The reaction mixture was concentrated under vacuum andobtained material was washed with Et₂O (2×15 mL) to afford compound2S-BK (3.5 g, 97%) as HCl salt.

¹H-NMR: (500 MHz, DMSO-d₆) (Rotamers): δ8.49 (br s, 3H), 8.15 (br s,1H), 5.14-5.10 (m, 1H), 4.26-4.22 (m, 1H), 3.97-3.95 (m, 1H), 3.59 (s,2H), 2.09 (s, 3H), 1.98 (s, 2H), 1.87-1.80 (m, 2H), 1.26 (d, 3H).

LCMS (ESI): 215.1 [M⁺+1].

Synthesis of tert-butyl ((2S,3R)-3-(benzyloxy)-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl) carbamate (2S-BL)

To a stirring solution of compound 2S-B (8 g, 25.8 mmol) in DCM (80 mL)were added N,N-diisopropylethylamine (11 mL, 87.4 mmol), pyrrolidine(2.5 mL, 35.4 mmol), followed by EDCI (7.39 g, 38.7 mmol), HOBT (5.2 g,38.7 mmol) at 0° C. and stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was diluted with water(20 mL). The separated organic layer was washed with saturated NaHCO₃solution (1×25 mL), followed by brine solution (1×30 mL). The separatedorganic layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to afford crude compound which was purified by columnchromatography eluting 1% MeOH/DCM to obtained compound 2S-BL (8 g, 86%)as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.34-7.24 (m, 5H), 6.57 (d, J=4.0 Hz, 1H),4.53, 4.44 (dd, J=12.0 Hz, 12.0 Hz, 2H), 4.32-4.28 (m, 1H), 3.74 (t,J=6.0 Hz, 1H), 3.58-3.53 (m, 1H), 3.42-3.38 (m, 1H), 3.28-3.24 (m, 2H),1.82-1.70 (m, 4H), 1.37 (s, 9H), 1.11 (d, J=6.4 Hz, 3H)

Mass (ESI): m/z 363.4 [M⁺+1].

Synthesis of (2S,3R)-2-amino-3-(benzyloxy)-1-(pyrrolidin-1-yl)butan-1-one (2S-BM)

To a stirring solution of compound 2S-BL (6 g, 16.52 mmol) in ethersaturated with HCl (30 mL) was added at 0° C. and stirred at RT for 2 h.After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure to afford crude compoundwhich was triturated with pentane (15 mL) to obtained compound 2S-BM (4g, 93%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.25 (s, 2H), 7.36-7.29 (m, 5H), 4.58,4.48 (dd, J=12.0, 11.5 Hz, 2H), 4.12 (t, J=4.5 Hz, 1H), 3.87 (t, J=5.5Hz, 1H), 3.60-3.57 (m, 1H), 3.37-3.33 (m, 3H), 1.78-1.70 (m, 4H), 1.22(d, J=6.5 Hz, 3H).

Mass (ESI): 263.3 [M⁺+1].

Synthesis oftert-Butyl2-((benzyloxy)methyl)-2-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)pyrrolidine-1-carboxylate (2S-1)

To a stirred solution of compound 2S-H (2.0 g, 5.97 mmol) in CH₂Cl₂ (10mL) was added DIPEA (2.6 mL, 14.92 mmol) followed by HATU (2.26 g, 5.94mmol) at 0° C. and stirred for 10 minutes. A solution of methyl2-amino-3-hydroxybutanoate hydrochloride (1 g, 5.97 mmol) in CH₂Cl₂ (10mL) was added to the reaction mixture at 0° C. The resultant reactionmixture was allowed to warm to RT and stirring was continued for another3 h. The volatiles were evaporated under reduced pressure. The obtainedresidue was diluted with water (25 mL) and extracted with CH₂Cl₂ (2×75mL). The organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure. The crude was purified by silica gel columnchromatography eluting with 40% EtOAc/Hexane to afford compound 2S-1(1.8 g, 67%) as a liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.38-7.31 (m, 5H), 5.16 (br s, 1H), 4.54(s, 2H), 4.28-4.26 (m, 1H), 4.17-4.12 (m, 1H), 3.84-3.82 (m, 1H), 3.62(s, 3H), 3.54-3.51 (m, 1H), 2.32-2.27 (m, 4H), 1.84-1.78 (m, 2H), 1.42(s, 9H), 1.06 (d, 3H);

LCMS m/z: 451.6 [M⁺+1]

Synthesis oftert-Butyl-2-(((2S,3R)-3-acetoxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-2-((benzyloxy)methyl)pyrrolidine-1-carboxylate (2S-2)

To a stirred solution of compound 2S-1 (1.0 g, 2.22 mmol) in CH₂Cl₂ (15mL) was added Et₃N (0.34 mL, 2.44 mmol) drop wise at 0° C. under inertatmosphere. To this Ac₂O (0.27 mL, 2.64 mmol) followed by DMAP (50 mg,0.40 mmol) was added at 0° C. and allowed to stir at RT for 2 h. Thereaction mixture was diluted with water and the aqueous layer wasextracted with CH₂Cl₂ (2×25 mL). The combined organic extracts weredried over anhydrous Na₂SO₄ and concentrated under reduced pressure toafford compound 2S-2 (0.8 g, 73%) as a liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.41-7.34 (m, 5H), 5.26-5.24 (m, 1H), 4.54(s, 2H), 4.06-3.97 (m, 1H), 3.78-3.72 (m, 1H), 3.62 (s, 3H), 3.52-3.49(m, 1H), 2.68 (s, 6H), 2.34-2.31 (m, 1H), 1.87 (s, 3H), 1.78-1.74 (m,2H), 1.42 (s, 6H), 1.14 (d, 3H).

Mass m/z: 493.8 [M⁺+1]

Synthesis oftert-Butyl-2-(((2S,3R)-3-acetoxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-2-(hydroxymethyl)pyrrolidine-1-carboxylate (2S-3)

To a stirred solution of compound 2S-2 (0.8 g, 1.62 mmol) in EtOAc (15mL) was added 10% Pd-C (0.15 g) and stirred at RT for 24 h under H₂atmosphere (balloon pressure). The reaction mixture was filtered througha celite pad and washed with EtOAc. The filtrate was concentrated underreduced pressure. The crude compound was purified by silica gel columnchromatography eluting with 40% EtOAc/hexane to afford compound 2S-3(0.5 g, 77%) as a liquid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.16 (br s, 1H), 5.78-5.74 (m, 1H), 5.23(d, J=9.5 Hz, 1H), 4.64-4.58 (m, 1H), 4.03-3.98 (m, 1H), 3.61 (s, 3H),3.52 (d, J=10.0 Hz, 1H), 3.43 (d, 0.1=6.5 Hz, 1H), 2.29-2.27 (m, 1H),1.96-1.94 (m, 4H), 1.74-1.68 (m, 2H), 1.38-1.30 (m, 9H), 1.14 (d, J=6.5Hz, 3H);

LCMS m/z: 403.6 [M⁺+1]

Synthesis oftert-Butyl-2-((2S,3R)-3-acetoxy-1-methoxy-1-oxobutan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate (2S-FNL-1)

To a stirred solution of compound 2S-3 (0.35 g, 0.87 mmol) in THF (15mL) was added PPh₃ (274 mg, 1.04 mmol) at RT and stirred for 30 minutesunder inert atmosphere. Then the reaction mixture was cooled 0° C., DTAD(0.22 g, 0.95 mmol) was added to the reaction mixture and allowed towarm to room temperature and stirring was continued for another 20 h. Itwas quenched with saturated citric acid and washed with saturated NaClsolution and extracted with EtOAc (2×20 mL). The combined organicextract were dried over anhydrous Na₂SO₄ and concentrated under reducedpressure. The obtained crude material was purified by silica gel columnchromatography eluting with 40% EtOAc/Hexane to afford NRX-1076(2S-FNL-1) (160 mg, 48%) as a liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 5.21-5.18 (m, 1H), 4.57 (d, 1H), 3.82 (d,1H), 3.64 (d, 3H), 3.42 (d, 2H), 3.24-3.21 (m, 1H), 2.14-2.11 (m, 2H),1.97 (s, 3H), 1.84-1.78 (m, 2H), 1.37 (s, 9H), 1.18 (d, 3H);

Mass m/z: 383.1 [M−1]

Synthesis of tert-butyl2-((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-2)

To a stirring solution of compound 2S-L (500 mg, 1.52 mmol) in CH₂Cl₂ (5mL) were added DIPEA (0.8 mL, 4.57 mmol), EDCI.HCl (350 mg, 1.82 mmol)followed by HOBt (280 mg, 1.82 mmol), NH₄Cl (161 mg, 3.04 mmol) at 0° C.and stirred for 16 h at RT. After consumption of the starting material(by TLC), the reaction mixture was diluted with water (10 mL) andextracted with CH₂Cl₂ (2×30 mL). The combined organic layer was washedwith citric acid solution (2×30 mL). The organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure.Obtained crude material was purified by silica gel column chromatographyeluting 2% MeOH/DCM to afford compound (2S-FNL-2) (200 mg, 40%) ascolorless liquid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.53 (s, 2H), 4.59 (s, 1H), 4.02 (s, 1H),3.77-3.70 (m, 2H), 3.62-3.53 (m, 2H), 3.46-3.33 (m, 1H), 2.17-2.03 (m,2H), 1.88-1.71 (m, 2H), 1.38 (s, 9H), 1.18 (d, J=6.5 Hz, 3H);

Mass (ESI): 328.3 [M⁺+1]

Synthesis of (2S,3R)-3-hydroxy-2-(1-oxo-2,5-diazaspiro[3,4]octan-2-yl)butanamide (2S-FNL-3)

To a stirring solution of compound (2S-FNL-2) (200 mg, 0.61 mmol) inCH₂Cl₂ (5 mL) was added TFA (0.5 mL, 6.1 mmol) at 0° C. and stirred atRT for 3 h. After completion of reaction (by TLC), the reaction mixturewas concentrated under reduced pressure to obtained crude compound whichwas triturated with n-pentane/diethylether (5 mL/5 mL) to affordcompound (2S-FNL-3) (100 mg) as white solid (TFA salt).

¹H-NMR: (400 MHz, D₂O): δ 4.33-4.29 (m, 2H), 4.09 (d, 1H), 3.95 (d, 1H),3.57-3.48 (m, 2H), 2.51-2.46 (m, 2H), 2.25-2.19 (m, 2H), 1.31 (d, 3H);

LCMS, m/z: 455 [21\e+1]

Synthesis of(2S,3R)-3-hydroxy-2-(5-isobutyryl-1-oxo-2,5-diazaspiro[3,4]octan-2-yl)butanamide (2S-FNL-4)

To a stirring solution of (2S-FNL-3) (500 mg (crude), 2.20 mmol) inCH₂Cl₂ (10 mL) was added TEA (1 mL, 7.70 mmol) followed by SM3 (256 mg,2.42 mmol) at 0° C. and stirred for 16 h at RT. After consumption of thestarting material (by TLC), the reaction mixture was diluted with water(10 mL) and extracted with CH₂Cl₂ (2×30 mL). The combined organic layerwas washed with citric acid solution (2×30 mL). The organic layer wasdried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure. Obtained crude material was purified by silica gel columnchromatography eluting 2% MeOH/DCM to afford (2S-FNL-4) (100 mg, 15.2%)as white solid.

¹H-NMR: (500 MHz, D₂O): δ 4.54-4.52 (m, 1H), 4.41-4.37 (m, 1H), 4.27 (d,J=3.6 Hz, 1H), 4.04 (t, J=6.5 Hz, 1H), 3.85-3.72 (m, 1H), 3.71-3.66 (m,1H), 2.92-2.87 (m, 1H), 2.38-2.27 (m, 2H), 2.12-2.05 (m, 2H), 1.30 (d,J=6.5 Hz, 3H), 1.14 (d, J=6.5 Hz, 6H);

Mass (ESI): 298.3 [M⁺+1]

Synthesis of(2S,3R)-2-(5-(1-benzyl-5-methyl-1H-1,2,3-triazole-4-carbonyl)-1-oxo-2,5-diazaspiro[3,4]octan-2-yl)-3-hydroxybutanamide(2S-FNL-5)

To a stirring solution of 2S-X2 (200 mg, 0.92 mmol) in CH₂Cl₂ (10 mL),DMF (0.1 mL) were added oxalyl chloride (0.16 mL, 1.84 mmol) at 0° C.The reaction mixture was warmed to RT and stirred for 2 h. The volatileswere evaporated under reduced pressure in presence of N₂ atmosphere toafford acid chloride (300 mg, crude). To a stirred solution of acidchloride (300 mg, crude) in DCM (5 mL) was added (2S-FNL-3) (220 mg,0.92 mmol), N,N-diisopropylethylamine (0.53 mL, 2.76 mmol) at 0° C. Theresulting reaction mixture was stirred at RT for 1 h. After consumptionof the starting material (by TLC), the reaction mixture was diluted withwater (10 mL) and extracted with CH₂Cl₂ (2×20 mL). Combined organicextracts were washed by brine solution (2×10 mL) and dried overanhydrous Na₂SO₄ concentrated under reduced pressure to obtain crudeproduct, which was purified by silica gel column chromatography elutingwith 3% McOH/CH₂Cl₂ to afford compound (2S-FNL-5) (200 mg, 48.6%) aspale brown solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.77 (s, 2H), 7.38-7.31 (m, 3H), 7.18 (d,J=7.5 Hz, 2H), 5.65 (s, 2H), 4.89 (d, J=5.5 Hz, 1H), 4.76 (d, J=4.0 Hz,1H), 4.07-3.91 (m, 4H), 3.62-3.48 (m, 1H), 2.39 (s, 3H), 2.26-2.12 (m,2H), 1.98-1.91 (m, 2H), 1.13 (d, J=6.5 Hz, 3H);

LCMS m/z: 427.6 [M⁺+1];

HPLC: 95.5% (both enantiomers)

Synthesis of tert-butyl2-((benzyloxy)methyl)-2-(((2S,3R)-3-hydroxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)pyrrolidine-1-carboxylate(2S-4)

To a stirring solution of compound 2S-H (50 g, 0.15 mol) in CH₂Cl₂ (500mL) was added methyl 2-amino-3-hydroxybutanoate (23.8 g, 0.18 mol),EDCI.HCl (34.2 g, 0.18 mol) followed by HOBt (24.1 g, 0.18 mol) andDIPEA (57.8 g, 0.45 mol) at RT and stirred for 2 h. After consumption ofthe starting material (by TLC), the reaction mixture was diluted withwater (250 mL) and extracted with CH₂Cl₂ (2×250 mL). The separatedorganic layers were washed with water, brine, dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure. Obtained crudematerial was purified by silica gel column chromatography eluting with50% EtOAc/Hexane to afford compound 2S-4 (53 g, 78.9%) as light greenliquid.

Synthesis of tert-butyl2-(((2S,3R)-3-acetoxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-2-((benzyloxy)methyl)pyrrolidine-1-carboxylate(2S-5)

To a stirring solution of compound 2S-4 (15 g, 33.3 mmol) in CH₂Cl₂ (150mL) was added DIPEA (6.4 g, 49.9 mmol) followed by acetic anhydride (4g, 39.9 mmol) and DMAP (408 mg, 3.33 mmol) at RT and stirred for 2 h.After consumption of the starting material (by TLC), the reactionmixture was diluted with water (100 mL) and extracted with CH₂Cl₂ (2×100mL). The separated organic layer was washed with brine, dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toafford compound 2S-5 (16 g, crude) as light brown liquid.

¹H-NMR: (400 MHz, CDCl₃): δ7.35 (d, 5H), 5.47-5.44 (m, 1H), 4.80 (dd,1H), 4.64-4.61 (m, 2H), 4.15-4.11 (m, 1H), 3.86-3.83 (m, 1H), 3.75 (s,4H), 3.54-3.50 (m, 2H), 2.42-3.38 (m, 1H), 1.91-1.85 (m, 5H), 1.45-1.41(m, 10H), 1.27 (d, 2H); LCMS (ESI): 492 [M⁺]

Synthesis of tert-butyl2-(((2S,3R)-3-acetoxy-1-methoxy-1-oxobutan-2-yl)carbamoyl)-2-(hydroxymethyl)pyrrolidine-1-carboxylate(2S-6)

To a stirring solution of compound 2S-5 (16 g, 32.5 mmol) in methanol(100 mL) and EtOAc (100 mL) was added 10% Pd on Charcoal (3 g) at RT andstirred for 4 h under H₂ atmosphere (balloon pressure). Afterconsumption of the starting material (by TLC), the reaction mixture wasfiltered through a pad of celite and the pad was washed with methanol.Obtained filtrate was concentrated under reduced pressure to affordcompound 2S-6 (10 g, crude) as brown thick syrup. This material wasdirectly used for the next step without further purification.

Synthesis of (Z)-tert-butyl2-(1-methoxy-1-oxobut-2-en-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-7)

To a stirring solution of compound 2S-6 (10 g, 24.8 mmol) in THF (50 mL)was added triphenylphosphine (13 g, 49.7 mmol) and DTAD (11.15 g, 37.3mmol). The reaction mixture was stirred at RT for 16 h. Afterconsumption of the starting material (by TLC), the reaction wasconcentrated under reduced pressure. The crude material was purified bysilica gel column chromatography eluting with 40% EtOAc/hexane to affordcompound 2S-7 (2 g, 24.8%).

Synthesis of tert-butyl2-(1,3-diamino-1-oxobutan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-6)

A solution of compound 2S-7 (2 g, 6.16 mmol) in methanole.NH₃ (50 mL)was stirred at RT for 4 h. After consumption of the starting material(by TLC), the reaction mixture was concentrated under reduced pressure.Obtained crude material was washed with Et₂O (25 mL) and n-pentane (25mL) to afford (2S-FNL-6) (0.35 g, 16.6%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.70 (br s, 1H), 7.07 (s, 1H), 3.85 (d,1H), 3.73 (d, 1H), 3.42-3.38 (m, 2H), 3.29-3.25 (m, 1H), 3.12-3.07 (m,1H), 2.09 (t, 2H), 1.95 (br s, 1H), 1.84-1.81 (m, 2H), 1.93 (s, 9H),1.12 (d, 1H), 0.99 (d, 2H); LCMS (ESI) m/z: 327.3 [M⁺+1]

Synthesis of 3-amino-2-(1-oxo-2,5-diazaspiro[3,4]octan-2-yl)butanamide(2S-FNL-7)

To a stirring solution of (2S-FNL-6) (0.25 g, 0.76 mmol) in CH₂Cl₂ (10mL) was added ether.HCl (5 mL) at RT and stirred for 4 h. To this wasadded 1,4-dioxane-HCl (5 mL) and stirring was continued for 2 h. Afterconsumption of starting material (by TLC), the solvent from the reactionwas removed under reduced pressure and obtained crude material waswashed with ACN (25 mL) and Et₂O (25 mL) to afford (2S-FNL-7) (0.11 g,63.5%) as an off-white solid.

¹H-NMR: (400 MHz, D₂O): δ 4.69-4.55 (m, 1H), 4.12-3.86 (m, 3H),3.62-3.51 (m, 2H), 2.56-2.23 (m, 2H), 2.25-2.21 (m, 2H), 1.52-1.43 (m,3H).

LCMS (ESI) m/z: 227.2 [M⁺+1];

UPLC (Purity): 97.96%

Synthesis of tert-butyl2-((2S,3R)-3-hydroxy-1-(isobutylamino)-1-oxobutan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-8)

To a stirring solution of compound 2S-L (300 mg, 0.91 mmol) in CH₂Cl₂(10 mL) were added DIPEA (354 mg, 2.74 mmol), EDCI.HCl (210 mg, 1.09mmol) followed by HOBt (165 mg, 1.09 mmol), isobutylamine (80 mg, 1.09mmol) at 0° C. and stirred for 16 h at RT. After consumption of thestarting material (by TLC), the reaction mixture was diluted with water(10 mL) and extracted with CH₂Cl₂ (2×30 mL). The combined organic layerwas washed with brine (2×30 mL), dried over anhydrous Na₂SO₄, filteredand concentrated under reduced pressure. Obtained crude material waspurified by silica gel column chromatography eluting 2% MeOH/DCM toafford compound 2S-8 (250 mg, 71.5%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.02 (t, J=5.2 Hz, 1H), 4.37 (s, 2H), 4.04(t, J=5.6 Hz, 1H), 4.00 (d, J=5.6 Hz, 1H), 3.77-3.72 (m, 1H), 3.61 (d,J=6.0 Hz, 1H), 3.44-3.38 (m, 1H), 2.98-2.84 (m, 3H), 2.19-2.08 (m, 3H),1.84-1.79 (m, 1H), 1.42 (s, 9H), 1.39 (d, J=5.5 Hz, 3H), 0.84 (d, J=6.4Hz, 6H);

Mass (ESI): m/z 384.4 [M⁺+1]

Synthesis of(2S,3R)-3-hydroxy-N-isobutyl-2-(1-oxo-2,5-diazaspiro[3,4]octan-2-yl)butanamide (2S-FNL-8)

To a stirring solution of compound 2S-8 (250 mg, 0.65 mmol) in CH₂Cl₂ (5mL) was added TFA (0.6 mL, 6.52 mmol) at 0° C. and stirred at RT for 3h. After completion of reaction (by TLC), the reaction mixture wasconcentrated under reduced pressure to obtained crude compound which wastriturated with n-pentane/diethylether (3×5 mL) to afford (2S-FNL-8)(125 mg, 67.9%) as an white solid (TFA salt).

¹H-NMR: (500 MHz, D₂O): δ 4.26 (s, 2H), 4.09 (t, J=8.0 Hz, 1H), 3.95 (t,J=7.5 Hz, 1H), 3.56-3.51 (m, 2H), 3.14-3.06 (m, 2H), 2.50-2.43 (m, 2H),2.25-2.21 (m, 2H), 1.85-1.82 (m, 1H), 1.30 (d, J=5.5 Hz, 3H), 0.96 (d,J=7.0 Hz, 6H);

Mass (ESI): m/z 284.3 [M⁺+1]

Synthesis of tert-butyl 2-((2S,3R)-1-((cyclobutylmethyl)amino)-3-hydroxy-1-oxobutan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-9)

To a stirring solution of compound 2S-L (500 mg, 1.52 mmol) in CH₂Cl₂(10 mL) were added DIPEA (0.8 mL, 4.57 mmol), EDCI.HCl (350 mg, 1.82mmol) followed by HOBt (280 mg, 1.82 mmol), cyclobutylamine (155 mg,1.82 mmol) at 0° C. and stirred for 16 h at RT. After consumption of thestarting material (by TLC), the reaction mixture was diluted with water(10 mL) and the separated organic layer was washed with citric acid(2×20 mL), brine (2×20 mL). The combined organic layers were dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure.Obtained crude material was purified by silica gel column chromatographyeluting 80% EtOAc/n-hexane to afford compound 2S-9 (250 mg, 41.5%) ascolorless syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.20 (t, J=11.5 Hz, 1H), 4.53 (s, 2H),4.03 (t, J=7.5 Hz, 1H), 3.88 (t, J=8.5 Hz, 2H), 3.41-3.33 (m, 2H),3.32-3.24 (m, 2H), 2.41-2.33 (m, 3H), 2.32-2.27 (m, 2H), 2.24-2.17 (m,2H), 2.10-1.90 (m, 2H), 1.68 (t, J=8.5 Hz, 2H), 1.40 (s, 9H), 1.18 (d,J=6.4 Hz, 3H);

Mass (ESI): m/z 396.4 [1\e+1]

Synthesis of(2S,3R)-N-(cyclobutylmethyl)-3-hydroxy-2-(1-oxo-2,5-diazaspiro[3,4]octan-2-yl)butanamide (2S-FNL-9)

To a stirring solution of compound 2S-9 (250 mg, 0.63 mmol) in CH₂Cl₂ (5mL) was added TFA (0.5 mL, 5.06 mmol) at 0° C. and stirred at RT for 3h. After completion of reaction (by TLC), the reaction mixture wasconcentrated under reduced pressure to obtained crude compound which wastriturated with diethylether (5 mL) to afford (2S-FNL-9) (90 mg, 48.3%)as hygroscopic white solid (TFA salt).

¹H-NMR: (500 MHz, D₂O): δ4.23 (s, 2H), 4.08 (t, J=7.0 Hz, 1H), 3.94 (t,J=8.5 Hz, 1H), 3.56-3.51 (m, 2H), 3.32-3.24 (m, 2H), 2.56-2.53 (m, 3H),2.48-2.43 (m, 2H), 2.25-2.21 (m, 2H), 2.07-1.88 (m, 2H), 1.71 (t, J=8.5Hz, 2H), 1.28 (d, J=6.4 Hz, 3H);

Mass (ESI): m/z 296.3 [M⁺+1];

Synthesis of tert-butyl2-((2S,3R)-1-(benzylamino)-3-hydroxy-1-oxobutan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-10)

To a stirring solution of compound 2S-L (1 g, 3.04 mmol) in CH₂Cl₂ (15mL) were added DIPEA (1.6 mL, 9.14 mmol), EDCI.HCl (700 mg, 3.66 mmol)followed by HOBT (560 mg, 3.66 mmol), benzylamine (325 mg, 3.04 mmol) at0° C. and stirred for 16 h at RT.

After consumption of the starting material (by TLC), the reactionmixture was diluted with water (20 mL) and extracted with CH₂Cl₂ (2×30mL). The combined organic layer was washed with citric acid solution(2×30 mL) and organic layer was dried over anhydrous Na₂SO₄, filteredand concentrated under reduced pressure. Obtained crude material waspurified by silica gel column chromatography eluting 5% MeOH/DCM toafford (2S-FNL-10) (800 mg, 63.5%) as white solid.

¹H-NMR: (400 MHz, CD₃OD): δ 7.38-7.20 (m, 5H), 4.62-4.60 (m, 1H),4.49-4.43 (m, 2H), 4.33-4.25 (m, 1H), 4.04 (d, J=5.6 Hz, 1H), 3.96-3.92(m, 1H), 3.51-3.45 (m, 1H), 3.43-3.31 (m, 1H), 2.31-2.21 (m, 2H),1.98-1.86 (m, 2H), 1.39 (s, 9H), 1.24-1.22 (m, 3H);

Mass (ESI): m/z 418.4 [M⁺+1];

HPLC: 91.8% (both isomers)

Synthesis of(2S,3R)-N-benzyl-3-hydroxy-2-(1-oxo-2,5-diazaspiro[3,4]octan-2-yl)butanamide (2S-FNL-11)

To a stirring solution of (2S-FNL-10) (700 mg, 1.67 mmol) in CH₂Cl₂ (10mL) was added TFA (1.9 mL, 16.7 mmol) at 0° C. and stirred at RT for 4h. After completion of reaction (by TLC), the reaction mixture wasconcentrated under reduced pressure to obtained crude compound which wastriturated with n-pentane/diethylether (5 mL/5 mL) to afford (2S-FNL-11)(400 mg, 75.6%) as an white solid (TFA salt).

¹H-NMR: (400 MHz, D₂O): δ 7.45-7.34 (m, 5H), 4.45 (s, 2H), 4.29-4.21 (m,2H), 4.06-3.85 (m, 2H), 3.52-3.47 (m, 2H), 2.45-2.35 (m, 2H), 2.22-2.16(m, 2H), 1.24-1.20 (m, 3H);

Mass (ESI): m/z 318.4 [M⁺±1]; HPLC: 89.1% (both isomers).

Synthesis of(2S,3R)-2-(5-acetyl-1-oxo-2,5-diazaspiro[3,4]octan-2-yl)-N-benzyl-3-hydroxybutanamide(2S-FNL-12)

To a stirring solution of (2S-FNL-11) (240 mg, 0.75 mmol) in CH₂Cl₂ (10mL) were added TEA (0.31 mL, 2.25 mmol) at RT. After added acetylchloride (0.1 mL, 0.9 mmol) slowly at 0° C. and stirred to RT for 2 h.After consumption of the starting material (by TLC), the reactionmixture was diluted with water (5 mL) and extracted with CH₂Cl₂ (2×20mL). The combined organic layer was washed with citric acid solution(1×20 mL), brine (1×20 mL). The separated organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure.Obtained crude material was purified by silica gel column chromatographyeluting with 2% MeOH/DCM to afford (2S-FNL-12) (90 mg, 33.4%) as anoff-white solid.

¹H-NMR: (400 MHz, CD₃OD): δ 7.32-7.20 (m, 5H), 4.58-4.55 (m, 1H),4.52-4.42 (m, 2H), 4.36-4.22 (m, 1H), 4.08-3.93 (m, 1H), 3.70-3.65 (m,2H), 3.64-3.53 (m, 2H), 2.32-2.22 (m, 2H), 2.20 (s, 3H), 2.04-1.95 (m,2H), 1.22-1.20 (m, 3H);

Mass (ESI): m/z 360.3 [M⁺+1];

HPLC: 97.5% (both isomers)

Synthesis of(2S,3R)-N-benzyl-3-hydroxy-2-(5-isobutyryl-1-oxo-2,5-diazaspiro[3,4]octan-2-yl)butanamide (NRX-2563) (2S-FNL-13)

To a stirring solution of (2S-FNL-11) (244 mg, 0.76 mmol) in CH₂Cl₂ (10mL) was added TEA (0.37 mL, 2.66 mmol) at 0° C. After added Int-F (89mg, 0.84 mmol) and stirred at RT for 2 h. After completion of reaction(by TLC), the reaction mixture was diluted with water (10 mL) andextracted with CH₂Cl₂ (2×20 mL). The combined organic layer was washedwith citric acid solution (2×30 mL) and organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure.Obtained crude material was purified by silica gel column chromatographyeluting 60% EtOAc/n-hexane to afford (2S-FNL-13) (150 mg, 51%) as anoff-white solid.

¹H-NMR: (400 MHz, CD₃OD): δ 7.35-7.20 (m, 5H), 4.82-4.49 (m, 1H),4.46-4.31 (m, 1H), 4.29-4.03 (m, 1H), 3.90-3.70 (m, 2H), 3.69-3.57 (m,2H), 3.46-3.31 (m, 1H), 2.77-2.73 (m, 1H), 2.28-2.21 (m, 2H), 2.06-1.97(m, 2H), 1.22 (d, J=6.8 Hz, 3H), 1.08-0.98 (m, 6H);

Mass (ESI): m/z 388.4 [M⁺+1];

HPLC: 95.2% (both isomers)

Synthesis of tert-butyl 2-((2S,3R)-1-(((1,2,4-oxadiazol-5-yl) methyl)amino)-3-hydroxy-1-oxobutan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-14)

To a stirring solution of compound 2S-L (600 mg, 1.82 mmol) in DMF (10mL) were added DIPEA (708 mg, 5.48 mmol), 2S-Q (290 mg, 1.82 mmol) HATU(761 mg, 2.00 mmol) at 0° C. and stirred for 16 h at RT. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with water (50 mL) and EtOAc (100 mL). The organic layer waswashed with water (2×50 mL) followed by brine solution (2×30 mL). Theorganic layer was dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure. Obtained crude material was purified by silicagel column chromatography eluting 2% MeOH/DCM. The obtained solid wastriturated with ether/n-pentane (5 mL/5 mL) to afford (2S-FNL-14) (100mg, 13.5%) as white solid.

¹H-NMR: (400 MHz, CD₃OD): δ 8.61 (s, 1H), 4.85 (s, 2H), 4.78-4.63 (m,1H), 4.59-4.55 (m, 1H), 4.30-4.25 (m, 1H), 3.52-3.46 (m, 2H), 3.43-3.29(m, 1H), 2.31-2.23 (m, 2H), 1.96-1.88 (m, 2H), 1.45 (s, 9H), 1.26-1.20(m, 3H);

Mass (ESI): m/z 410.4 [M⁺+1];

HPLC: 98.14% (both isomers)

Synthesis of tert-butyl 2-((2S,3R)-1-(((1,3,4-oxadiazol-2-yl) methyl)amino)-3-hydroxy-1-oxobutan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-15)

To a stirring solution of compound 2S-L (1 g, 3.04 mmol) in DMF (10 mL)were added DIPEA (1.58 mL, 9.12 mmol), BOP reagent (2.01 g, 4.56 mmol)followed by 2S-U (496 mg, 3.64 mmol) at 0° C. and stirred for 16 h atRT. After consumption of the starting material (by TLC), the reactionmixture was diluted with water (100 mL) and extracted with EtOAc (2×30mL). The combined organic layer was washed with brine solution (2×50 mL)and organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. Obtained crude material waspurified by silica gel column chromatography eluting 80% EtOAc/n-hexanefollowed by preparative HPLC purification to afford (2S-FNL-15) (67 mg,5.4%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 10.92 (s, 1H), 7.83 (s, 1H), 4.97-4.88 (m,2H), 4.07 (d, J=7.2 Hz, 2H), 3.83-3.65 (m, 2H), 3.57-3.40 (m, 1H),3.38-3.25 (m, 2H), 2.15-2.01 (m, 2H), 1.83-1.80 (m, 2H), 1.40 (s, 9H),1.22 (d, J=6.4 Hz, 3H)

Mass (ESI): m/z 410.4 [M⁺+1];

HPLC: 90.6%

Synthesis of (2S,3R)-N-((1,3,4-oxadiazol-2-yl)methyl)-3-hydroxy-2-(1-oxo-2,5-diazaspiro[3,4]octan-2-yl) butanamide(2S-FNL-16)

To a stirring solution of compound (2S-FNL-15) (70 mg, 0.71 mmol) inCH₂Cl₂ (5 mL) was added TFA (195 mg, 1.71 mmol) at 0° C. and stirred atRT for 1 h. After completion of reaction (by TLC), the reaction mixturewas concentrated under reduced pressure to obtained crude compound whichwas triturated with n-pentane/diethylether (5 mL/5 mL) to affordcompound (2S-FNL-16) (60 mg, 84.5%) as an off-white solid (TFA salt).

¹H-NMR: (400 MHz, D₂O): δ7.83 (s, 1H), 5.20-5.10 (m, 1H), 4.80 (s, 1H),4.39-4.30 (m, 2H), 4.13-4.04 (m, 2H), 3.53-3.48 (m, 2H), 2.44-2.41 (m,2H), 2.21-2.16 (m, 2H), 1.31 (d, J=6.4 Hz, 3H); Mass (ESI): m/z 310.1[M⁺+1];

HPLC: 90.99%

Synthesis of benzyl(2S,3R)-3-(benzyloxy)-2-(1-oxo-2,5-diazaspiro[3,4]octan-2-yl) butanoate(8)

To a stirring solution of compound 2S-K (800 mg, 1.57 mmol) in DCM (10mL) was added TFA (1.2 mL) at 0° C. and stirred at RT for 2 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasconcentrated under reduced pressure to afford compound 2S-10 (500 mg,78%) as an off-white solid (HCl salt) was used directly for next step.

¹H-NMR: (500 MHz, D₂O): δ 7.48 (m, 5H), 7.24-7.21 (m, 5H), 5.29 (s, 2H),4.96 (s, 2H), 4.80-4.62 (m, 1H), 4.29-4.18 (m, 2H), 4.01-3.89 (m, 1H),3.52-3.46 (m, 2H), 2.43-2.38 (m, 2H), 2.24-2.14 (m, 2H), 1.35-1.28 (m,3H);

LCMS: 408 [M⁺+1]

Synthesis of benzyl(2S,3R)-2-(5-acetyl-1-oxo-2,5-diazaspiro[3,4]octan-2-yl)-3-(benzyloxy)butanoate (2S-11)

To a stirring solution of compound 2S-10 (500 mg, 1.22 mmol) in DCM (5mL) was added TEA (0.46 mL, 3.36 mmol) followed by acetyl chloride (0.1mL, 1.47 mmol) at 0° C. and stirred at RT for 2 h. After consumption ofthe starting material (by TLC), reaction mixture was diluted with water(10 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. Obtained crude material waspurified by silica gel column chromatography eluting 2% MeOH/DCM toafford compound 2S-11 (300 mg, 54.5%) as white solid.

¹H-NMR: (400 MHz, CD₃OD): 7.36-7.29 (m, 5H), 7.26-7.16 (m, 5H), 5.13 (s,2H), 4.59 (s, 2H), 4.32-4.29 (m, 2H), 4.16-4.13 (m, 1H), 3.65-3.61 (m,1H), 3.60-3.46 (m, 2H), 2.21-2.09 (m, 2H), 2.02 (s, 3H), 2.01-1.91 (m,2H), 1.21 (d, J=6.4 Hz, 3H); LCMS: 451.3 [M⁺+1]

Synthesis of(2S,3R)-2-(5-acetyl-1-oxo-2,5-diazaspiro[3,4]octan-2-yl)-3-hydroxybutanoicacid (2S-12)

To a stirring solution of compound 2S-11 (1 g, 2.22 mmol) in methanol(30 mL) was added 10% Pd/C (500 mg) at RT and stirred for 24 h under H₂atmosphere. After consumption of the starting material (by TLC), thereaction mixture was filtered through a pad of celite and the pad waswashed with methanol (20 mL). Obtained filtrate was concentrated underreduced pressure to afford compound 2S-12 (500 mg, 83.3%) as anoff-white solid.

¹H-NMR: (400 MHz, CD₃OD): δ 4.35-4.30 (m, 1H), 4.29-4.17 (m, 1H),4.09-4.04 (m, 1H), 3.76-3.67 (m, 1H), 3.59-3.48 (m, 1H), 3.34-3.31 (m,1H), 2.29-2.24 (m, 2H), 2.15 (s, 3H), 2.04-1.96 (m, 2H), 1.28 (d, J=6.4Hz, 3H);

LCMS m/z: 270.4 [M⁺+1]

Synthesis of(2S,3R)-2-(5-acetyl-1-oxo-2,5-diazaspiro[3,4]octan-2-yl)-3-hydroxy-N-(pyrimidin-2-ylmethyl)butanamide (2S-FNL-17)

To a stirring solution of compound 2S-12 (700 mg, 2.59 mmol) in DCM (15mL) were added DIPEA (1.35 mL, 7.77 mmol), 2S-Y (410 mg, 2.84 mmol),EDCI (593 mg, 3.1 mmol) followed by HOBT (474 mg, 3.1 mmol) at 0° C. andstirred for 16 h at RT. After consumption of the starting material (byTLC), the reaction mixture was diluted with water (40 mL). The organiclayer was dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure. Obtained crude material was purified by silica gelcolumn chromatography eluting 3% MeOH/DCM to afford (2S-FNL-17) (100 mg,10.7%) as white solid.

¹H-NMR: (400 MHz, D₂O): δ 8.78 (d, J=5.2 Hz, 2H), 7.49 (t, J=5.2 Hz,1H), 4.79 (s, 2H), 4.55-4.47 (m, 1H), 4.40-4.37 (m, 2H), 3.79-3.56 (m,3H), 2.37-2.26 (m, 2H), 2.14-2.03 (m, 2H), 2.01 (s, 3H), 1.28 (d, J=6.4Hz, 3H);

Mass (ESI): m/z 362.4 [M¹+1];

HPLC: 92.3% (both isomers)

Synthesis of tert-butyl2-((2S,3R)-3-hydroxy-1-oxo-1-((pyrimidin-2-ylmethyl) amino)butan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate (2S-FNL-18)

To a stirring solution of compound 2S-L (1 g, 3.04 mmol) in CH₂Cl₂ (30mL) were added DIPEA (1.63 mL, 9.14 mmol), EDCI.HCl (696 mg, 3.64 mmol)followed by HOBT (558 mg, 3.64 mmol), 2S-Y (241 mg, 3.34 mmol) at 0° C.and stirred for 16 h at RT. After consumption of the starting material(by TLC), the reaction mixture was diluted with water (30 mL). Theorganic layer was washed with citric acid solution (2×30 mL) followed bybrine solution (2×25 mL). The organic layer was dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure. Obtained crudematerial was purified by silica gel column chromatography eluting 5%MeOH/DCM to afford (2S-FNL-18) (800 mg, 63%) as white solid.

¹H-NMR: (400 MHz, CD₃OD): δ 8.72 (t, J=4.8 Hz, 2H), 7.36 (t, J=4.8 Hz,1H), 4.81-4.76 (m, 1H), 4.62-4.49 (m, 1H), 4.34-4.29 (m, 1H), 4.18-4.03(m, 2H), 3.56 (d, J=5.6 Hz, 2H), 3.52-3.46 (m, 1H), 2.30-2.25 (m, 2H),1.97-1.88 (m, 2H), 1.46 (s, 9H), 1.31-1.28 (m, 3H);

Mass (ESI): m/z 420.4 [M′+1];

HPLC: 99.6% (both isomers)

Synthesis of(2S,3R)-3-hydroxy-2-(1-oxo-2,5-diazaspiro[3,4]octan-2-yl)-N-(pyrimidin-2-ylmethyl)butanamide (2S-FNL-19)

To a stirring solution of (2S-FNL-18) (280 mg, 0.66 mmol) in CH₂Cl₂ (5mL) was added TFA (0.3 mL, 4.0 mmol) at 0° C. and stirred at RT for 4 h.After completion of reaction (by TLC), the reaction mixture wasconcentrated under reduced pressure to obtained crude compound that wastriturated with n-pentane/diethyl ether (5 mL/5 mL) to afford(2S-FNL-19) (95 mg, 44.6%) as an white solid (TFA salt).

¹H-NMR: (500 MHz, D₂O): δ 8.81 (d, J=4.5 Hz, 2H), 7.53 (t, J=5.0 Hz,1H), 4.80-4.65 (m, 2H), 4.46 (d, J=6.0 Hz, 1H), 4.36-4.31 (m, 2H), 4.10(d, J=7.5 Hz, 1H), 3.95 (t, J=8.0 Hz, 1H), 3.58-3.49 (m, 1H), 2.51-2.40(m, 2H), 2.26-2.17 (m, 2H), 1.34 (d, J=6.0 Hz, 3H);

Mass (ESI): m/z 320.3 [M⁺+1]

Synthesis of(2S,3R)-3-hydroxy-2-(5-isobutyryl-1-oxo-2,5-diazaspiro[3,4]octan-2-yl)-N-(pyrimidin-2-ylmethyl)butanamide (2S-FNL-20)

To a stirring solution of (2S-FNL-19) (300 mg, 0.94 mmol) in CH₂Cl₂ (5mL) was added TEA (0.4 mL, 2.82 mmol) followed by SM-4 (120 mg, 1.12mmol) at 0° C. and stirred at RT for 2 h. After completion of reaction(by TLC), the reaction mixture was diluted with water (10 mL) andextracted with CH₂Cl₂ (2×20 mL). The combined organic layer was driedover anhydrous Na₂SO₄, filtered and concentrated under reduced pressure.Obtained crude material was purified by silica gel column chromatographyeluting 2% MeOH/DCM to afford (2S-FNL-20) (100 mg, 27.3%) as whitesolid.

¹H-NMR: (400 MHz, CD₃OD): δ 8.73 (t, J=5.2 Hz, 2H), 7.36 (t, J=4.8 Hz,1H), 4.83-4.55 (m, 1H), 4.51-4.29 (m, 3H), 4.21-4.02 (m, 1H), 3.75-3.69(m, 1H), 3.64-3.60 (m, 1H), 3.31-3.30 (m, 1H), 2.79-2.72 (m, 1H),2.28-2.25 (m, 2H), 2.08-1.97 (m, 2H), 1.31 (d, J=6.4 Hz, 3H), 1.07-1.02(m, 6H).

Mass (ESI): m/z 390.4 [M⁺+1],

HPLC: 97.75%

tert-Butyl2-(((S)-3-((tert-butyldiphenylsilyl)oxy)-1-methoxy-1-oxopropan-2-yl)carbamoyl)-2-(hydroxymethyl)pyrrolidine-1-carboxylate(2S-13):

To a stirring solution of compound 2S-I (11 g, 44.89 mmol) in CH₂Cl₂(110 mL) was added compound 2S-AJ (16.07 g, 44.89 mmol), HATU (20.4 g,53.68 mmol) followed by DIPEA (17.37 g, 0.13 mol) at RT and stirred for10 h. After consumption of the starting material (by TLC), the reactionmixture was diluted with water (100 mL) and extracted with CH₂Cl₂ (2×100mL). The separated organic layer was washed with brine, dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure.Obtained crude material was purified by silica gel column chromatographyto afford compound 2S-13 (16 g, 61%) as yellow liquid.

¹H-NMR: (500 MHz, CDCl₃): δ 7.58-7.37 (m, 10H), 4.67 (s, 1H), 4.12-4.08(m, 2H), 3.93 (s, 1H), 3.75 (s, 3H), 3.72-3.64 (m, 2H), 2.8 (s, 1H),2.35 (s, 1H), 2.04 (s, 1H), 1.98-1.82 (m, 3H), 1.25 (s, 9H), 1.03 (s,9H);

Mass (ESI): m/z 583.5 [M⁺+1].

Synthesis of tert-butyl2-((S)-3-((tert-butyldiphenylsilyl)oxy)-1-methoxy-1-oxopropan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-14)

To a stirring solution of compound 2S-13 (1.6 g, 2.73 mmol) in THF (20mL) was added triphenylphosphine (0.994 g, 4.10 mmol) and DTAD (0.788 g,3.00 mmol). The reaction mixture was stirred at RT for 8 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with water and extracted with EtOAc (2×30 mL). The separatedorganic layer was dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure. The crude material was purified by silica gelcolumn chromatography to afford compound 2S-14 (0.8 g, 51%) as yellowsticky compound.

¹H-NMR: (400 MHz, CDCl₃): δ 7.63-7.58 (m, 4H), 7.45-7.30 (m, 6H), 4.1(s, 3H), 3.80-3.67 (m, 4H), 3.56-3.44 (m, 3H), 2.04-1.95 (m, 4H), 1.59(s, 9H), 1.04 (s, 9H).

Mass (ESI): m/z 567.4 [M⁺+1]

Synthesis of tert-butyl2-((S)-1-amino-3-((tert-butyldiphenylsilyl)oxy)-1-oxopropan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-15)

To a stirring solution of compound 2S-14 (6 g) in methanol (50 mL) wasadded methanolic ammonia (50 mL)) at 0° C. and stirred for 12 h at RT.After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure and purified the cruderesidue by silica gel column chromatography eluting 40% EtOAc:hexane toafford compound-2S-15 (1 g, 17%) as an pale yellow solid.

¹H-NMR: (400 MHz, CDCl₃): δ 8.27 (s, 1H), 7.67-7.63 (m, 4H), 7.45-7.36(m, 7H), 5.37 (s, 1H), 4.56-4.54 (m, 1H), 3.82 (d, J=5.2 Hz, 1H), 3.44(t, J=7.6 Hz, 2H), 3.35 (d, J=5.2 Hz, 1H), 3.21 (s, 1H), 2.09-2.06 (m,2H), 2.03 (d, J=4.8 Hz, 2H), 1.44 (s, 9H), 1.08 (s, 9H).

LCMS (M/Z) m/z: 214 [M⁺+1].

Synthesis of tert-butyl2-((S)-1-amino-3-hydroxy-1-oxopropan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-16)

To a stirring solution of compound 2S-15 (1 g, 1.81 mmol) in THF (10 mL)was added TBAF (0.943 g, 3.62 mmol) at 0° C. and the reaction mixturewas slowly warmed to RT and stirred for 2 h. After consumption of thestarting material (by TLC), the reaction mixture was diluted with water(5 mL) and extracted with EtOAc (2×15 mL). The separated organic layerwas washed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. Obtained crude material waspurified by silica gel column chromatography by eluting 3% MeOH:DCM toafford 2S-16 (0.13 g, 23%) as white solid.

¹H-NMR: (400 MHz, D₂O): δ 4.53 (t, J=6.8 Hz, 1H), 4.03 (d, J=4.8, 1H),3.96-3.91 (m, 2H), 3.85 (t, J=5.8 Hz, 1H), 3.82 (s, 2H), 2.30 (t, J=4Hz, 2H), 2.15-1.82 (m, 2H), 1.49 (s, 9H).

LCMS (M/Z) m/z: 314.2 [M⁺+1]

Synthesis of(2S)-3-hydroxy-2-(1-oxo-2,5-diazaspiro[3,4]octan-2-yl)propanamide(2S-FNL-21)

To a stirring solution of 2S-16 (0.13 g, 0.415 mmol) in CH₂Cl₂ (3 mL)was added TFA (1 mL) at 0° C. and stirred at RT for 2 h. The reactionmixture was concentrated under reduced pressure to afford (2S-FNL-21)(100 mg, crude) as TFA salt.

¹H-NMR: (400 MHz, D₂O): δ 4.58 (t, J=5.8 Hz, 1H), 4.09-4.03 (m, 3H),3.92 (d, J=7.2 Hz, 1H), 3.57-3.52 (m, 2H), 2.55-2.41 (m, 2H), 2.28-2.19(m, 2H);

LCMS (M/Z) m/z: 214[M⁺+1].

Synthesis of tert-butyl2-((S)-1-amino-3-hydroxy-1-oxopropan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-22)

To a stirring solution of compound 2S-AF (250 mg, 0.79 mmol) in DCM (10mL) were added DIPEA (0.5 mL, 2.38 mmol), EDCI (181 mg, 0.94 mmol), HOBT(127 mg, 0.94 mmol) followed by NH₄Cl (84.5 mg, 1.58 mmol) at 0° C. andstirred to RT for 12 h. After consumption of the starting material (byTLC), the reaction mixture was diluted with water (20 mL) and washedwith citric acid (1×30 mL) followed by brine solution (1×30 mL). Theorganic layer was dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure. Obtained crude material was purified by silicagel column chromatography eluting with 5% MeOH/DCM to afford (2S-FNL-22)(150 mg, 60.7%) as yellow thick syrup.

¹H-NMR: (400 MHz, CD₃OD): δ 4.13-4.07 (m, 2H), 3.96-3.88 (m, 1H),3.87-3.77 (m, 1H), 3.63-3.47 (m, 2H), 3.44-3.30 (m, 1H), 2.31-2.26 (m,2H), 1.97-1.88 (m, 2H), 1.47 (s, 9H); LCMS (ESI): m/z 314.3 [M⁺+1];

HPLC: 98.38%

Synthesis of tert-butyl 2-((S)-3-hydroxy-1-oxo-1-((pyrimidin-2-ylmethyl)amino) propan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-23)

To a stirring solution of compound 2S-AF (1.3 g, 4.14 mmol) in DCM (25mL) were added DIPEA (2.15 mL, 12.42 mmol), HOBT (760 mg, 4.96 mmol),EDCI (1 g, 4.96 mmol) followed by 2S-Y (715 mg, 4.96 mmol) at 0° C. andstirred for 16 h at RT. After consumption of the starting material (byTLC), the reaction mixture was diluted with water (50 mL). The organiclayer was washed with citric acid (1×30 mL) followed by bicarbonatesolution (1×30 mL). The organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure. Obtained crudematerial was purified by silica gel column chromatography eluting with2% MeOH/DCM to afford (2S-FNL-23) (800 mg, 50%) as white solid.

¹H-NMR: (400 MHz, CD₃OD): δ 8.75-8.71 (m, 2H), 7.37-7.34 (m, 1H),4.66-4.49 (m, 2H), 4.27-4.24 (m, 1H), 4.19-4.14 (m, 1H), 4.03-3.99 (m,1H), 3.97-3.92 (m, 1H), 3.66-3.54 (m, 1H), 3.49-3.45 (m, 1H), 3.40-3.36(m, 1H), 2.32-2.27 (m, 2H), 1.97-1.88 (m, 2H), 1.47 (s, 9H);

Mass (ESI): m/z 406.4 [M⁺+1].

HPLC: 97.1%

Synthesis of(2S)-3-hydroxy-2-(1-oxo-2,5-diazaspiro[3,4]octan-2-yl)-N-(pyrimidin-2-ylmethyl)propanamide (2S-FNL-24)

To a stirring solution of compound (2S-FNL-23) (350 mg, 0.86 mmol) inDCM (5 mL) was added TFA (985 mg, 0.86 mmol) at 0° C. and stirred to RTfor 3 h. The reaction mixture was brought to RT and concentrated undervacuum to afford (2S-FNL-24) (250 mg, 95.4%) as white solid.

¹H-NMR: (400 MHz, D₂O): δ 8.84 (d, J=5.2 Hz, 2H), 7.55 (t, J=4.8 Hz,1H), 4.90-4.67 (m, 3H), 4.10-4.06 (m, 3H), 3.94-3.92 (m, 1H), 3.57-3.51(m, 2H), 2.54-2.43 (m, 2H), 2.28-2.19 (m, 2H);

LCMS: m/z 306.4 [M⁺⁺1];

HPLC: 90.07%.

Synthesis of(2S)-3-hydroxy-2-(5-isobutyryl-1-oxo-2,5-diazaspiro[3,4]octan-2-yl)-N-(pyrimidin-2-ylmethyl)propanamide (2S-FNL-25)

To a stirring solution of compound (2S-FNL-24) (500 mg, 1.63 mmol) inDCM (5 mL) was added TEA (0.7 mL, 4.91 mmol) at 0° C. After added Int-F(207 mg, 1.95 mmol) slowly and stirred to RT for 3 h. After consumptionof the starting material (by TLC), the reaction mixture was diluted withwater (20 mL). The organic layer was washed with citric acid (1×30 mL)followed by brine solution (1×30 mL). The organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure.Obtained crude material was purified by silica gel column chromatographyeluting with 5% MeOH/DCM to afford (2S-FNL-25) (100 mg, 16.3%) as whitesolid.

¹H-NMR: (400 MHz, CD₃OD): δ 8.73 (t, J=4.8 Hz, 2H), 7.37 (d, J=5.2 Hz,1H), 4.56-4.51 (m, 2H), 4.32-4.29 (m, 1H), 4.17-4.12 (m, 1H), 4.05-3.98(m, 2H), 3.74-3.68 (m, 1H), 3.63-3.58 (m, 1H), 3.57-3.51 (m, 1H),2.77-2.69 (m, 1H), 2.31-2.26 (m, 2H), 2.08-1.95 (m, 2H), 1.05-0.98 (m,6H);

LCMS: m/z 376.4 [M⁺+1];

HPLC: 89.6% (both isomers)

Synthesis of tert-butyl2-(((2S,3R)-3-acetoxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-1)carbamoyl)-2-((benzyloxy)methyl)pyrrolidine-1-carboxylate(2S-17)

To a stirring solution of compound 2S-BK (1 g, 2.90 mmol) in DMF (8 mL)was added EDCI.HCl (0.63 g, 3.28 mmol) followed by HOBt (0.44 g, 3.28mmol) at 0° C. After being stirred for 5 min, DIPEA (1.3 mL, 7.46 mmol)followed by compound 2S-H (0.74 g, 3.58 mmol) was added to the reactionmixture and stirring was continued for another 16 h at RT. The reactionmixture was washed with water and extracted with EtOAc (2×500 mL). Theorganic layer was washed with brine, dried over anhydrous Na₂SO₄ andconcentrated under vacuum. The crude was purified by columnchromatography to afford compound 2S-17 (0.6 g, 38%).

¹H-NMR: (400 MHz, CDCl₃) (Rotamers): δ7.34 (s, 5H), 5.37-5.34 (m, 1H),4.84-4.80 (m, 1H), 4.72-4.65 (m, 2H), 4.09-4.02 (m, 1H), 3.91-3.87 (m,1H), 3.65-3.61 (m, 3H), 3.52-3.46 (m, 3H), 2.41 (br s, 1H), 2.22-2.15(m, 1H), 1.98 (d, 5H), 1.87-1.84 (m, 4H), 1.50-1.42 (m, 9H).

LCMS m/z: 532 [M⁺+1].

Synthesis of tert-butyl2-(((2S,3R)-3-acetoxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-1)carbamoyl)-2-(hydroxymethyl)pyrrolidine-1-carboxylate(2S-18)

To a stirring solution of compound 2S-17 (4.5 g, 8.40 mmol) in MeOH (40mL) was added wet 10% Pd/C (1.5 g) under inert atmosphere and stirredfor 4 h under H₂ atmosphere (balloon pressure). The reaction mixture wasfiltered through celite pad and concentrated under reduced pressure toafford compound 2S-18 (3.0 g, 81%).

LCMS m/z: 442.5 [M⁺+1].

Synthesis of tert-butyl2-((2S,3R)-3-acetoxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-19)

To a stirring solution of compound 2S-18 (3 g, 6.70 mmol) in THF (25 mL)was added triphenylphosphine (2 g, 7.40 mmol) followed by DTAD (2.5 g,10.2 mmol). The reaction mixture was stirred at RT for 16 h. Afterconsumption of the starting material (by TLC), the reaction wasconcentrated under reduced pressure. The crude material was purified bysilica gel column chromatography eluting with 10% MeOH/CH₂Cl₂ to affordcompound 2S-19 (1.2 g with TPPO, 43%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 5.25-5.19 (m, 1H), 4.65 (d, 1H), 3.61-3.57(m, 3H), 3.47-3.42 (m, 2H), 3.41-3.25 (m, 4H), 2.05 (s, 4H), 1.95-1.71(m, 7H), 1.42 (s, 10H).

LCMS m/z: 424.4 [M⁺+1].

Synthesis of tert-butyl 2-((2S,3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate (2S-20)

A solution of compound 2S-19 (0.15 g, 0.41 mmol) in aqueous NH₃ (2 mL)was stirred at RT for 4 h. After consumption of the starting material(by TLC), the reaction diluted with CH₂Cl₂ (75 mL). The separatedorganic layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to afford compound 2S-20 (0.1 g, 76%).

LCMS m/z: 382 [M⁺+1].

Synthesis of2-((2S,3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-2,5diazaspiro[3,4]octan-1-one. (2S-FNL-26)

To a stirring solution of compound 2S-20 (0.2 g, 0.63 mmol) in CH₂Cl₂ (2mL) was added TFA (0.3 mL) at 0° C. and stirred at RT for 1 h. Thereaction mixture was concentrated under vacuum. Obtained residue wasdiluted with water and extracted with CH₂Cl₂ (2×25 mL). The separatedorganic layer was dried over anhydrous Na₂SO₄, filtered and concentratedunder vacuum to afford (2S-FNL-26) (0.2 g, 80%) as TFA salt.

¹H-NMR: (400 MHz, D₂O): δ 4.64 (t, 1H), 4.25-4.21 (m, 1H), 4.09 (d, 1H),3.99-3.87 (m, 1H), 3.70 (t, 2H), 3.55-3.47 (m, 5H), 2.52-2.34 (m, 2H),2.25-2.22 (m, 2H), 2.08-1.98 (m, 5H), 1.25 (t, 3H).

LCMS (ESI) m/z: 282.4 [M⁺+1].

Synthesis of(2R,3S)-4-oxo-3-(1-oxo-2,5-diazaspiro[3,4]octan-2-yl)-4-pyrrolidin-1-yl)butan-2-yl acetate (2S-FNL-27)

A stirring solution of compound 2S-19 (0.4 g, 0.94 mmol) in1,4-dioxane/HCl (5 mL) was cooled to 0° C. and stirred at RT for 1 h.After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure. Obtained crude materialwas washed with n-pentane followed by EtOAc to afford (2S-FNL-27) (0.22g, 65%).

¹H-NMR: (400 MHz, D₂O): δ 4.62 (d, 1H), 4.41-4.29 (m, 2H), 4.24 (d, 1H),3.89-3.77 (m, 3H), 3.54-3.49 (m, 3H), 2.57-2.52 (m, 1H), 2.49 (s, 3H),2.42-2.00 (m, 8H), 1.30 (d, 3H).

LCMS m/z: 324.3 [M⁺+1].

HPLC Purity: 99.37%.

Synthesis of tert-butyl2-((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)-6-methyl-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-21)

To a stirring solution of compound 2S-AQ (480 mg, 1.40 mmol) in CH₂Cl₂(15 mL) were added DIPEA (543 mg, 4.20 mmol), EDCI.HCl (382 mg, 2.0mmol) followed by HOBt (280 mg, 2.0 mmol), NH₄Cl (111 mg, 2.0 mmol) at0° C. and stirred at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was diluted with water (20 mL)and extracted with CH₂Cl₂ (2×30 mL). The combined organic layer waswashed with brine (2×50 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. Obtained crude material waspurified by silica gel column chromatography eluting 2% MeOH/DCM toafford compound 2S-21 (150 mg, 31%) as colorless thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ5.00-4.88 (m, 1H), 4.05-3.94 (m, 4H), 3.37(t, J=10.5 Hz, 2H), 2.10-1.93 (m, 4H), 1.45 (s, 9H), 1.39-1.27 (m, 1H),1.24-1.16 (m, 6H);

Mass (ESI): m/z 364.3 [M⁺+Na]

Synthesis of(2S,3R)-3-hydroxy-2-(1-methyl-3-oxo-2,5-diazaspiro[3,4]octan-2-yl)butanamide (2S-FNL-28)

To a stirring solution of compound 2S-21 (150 mg, 0.43 mmol) in CH₂Cl₂(5 mL) was added TFA (0.4 mL, 4.39 mmol) at 0° C. and stirred at RT for2 h. After completion of reaction (by TLC), the reaction mixture wasconcentrated under reduced pressure to obtained crude compound which wastriturated with diethylether/n-pentane (5 mL/5 mL) to afford (2S-FNL-28)(100 mg, 65.7%) as sticky solid (TFA salt). HPLC (purity): 99.7%

¹H-NMR: (400 MHz, D₂O): δ4.50-4.46 (m, 3H), 3.63-3.49 (m, 2H), 2.56-2.49(m, 2H), 2.35-2.29 (m, 2H), 1.57 (d, J=6.8 Hz, 3H), 1.36 (d, J=6.0 Hz,3H);

Mass (ESI): m/z 483.1 [2M⁺+1]

Synthesis of tert-butyl2-((2R,3S)-3-hydroxy-1-oxo-1-((pyrimidin-2-ylmethyl)amino)butan-2-yl)-1-methyl-3-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-29)

To a stirring solution of compound 2S-AQ (500 mg, 1.46 mmol) in CH₂Cl₂(15 mL) were added DIPEA (0.76 mL, 4.38 mmol), EDCI.HCl (334 mg, 1.75mmol), HOBt (334 mg, 1.75 mmol) followed by 2S-Y (252 mg, 1.75 mmol) at0° C. and stirred at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was diluted with water (20 mL)and extracted with CH₂Cl₂ (2×30 mL). The combined organic layer waswashed with citric acid solution (20 mL), NaHCO3 (1×30 mL) followed bybrine (1×50 mL). The organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure. Obtained crudematerial was purified by silica gel column chromatography eluting 2%MeOH/DCM to afford (2S-FNL-29) (200 mg, 31.6%) as white solid.

¹H-NMR: (400 MHz, CD₃OD): δ 8.74-8.70 (m, 2H), 7.37-7.32 (m, 1H),4.72-4.43 (m, 3H), 4.24-4.14 (m, 1H), 4.10-3.88 (m, 2H), 3.52-3.36 (m,2H), 2.22-2.19 (m, 2H), 2.01-1.94 (m, 1H), 1.88-1.79 (m, 1H), 1.45-1.41(m, 3H), 1.40 (s, 9H), 1.29-1.26 (m, 3H),

Mass (ESI): 434.5 [M⁺+1], HPLC: 92.8%

Synthesis of(2S,3R)-3-hydroxy-2-(1-methyl-3-oxo-2,5-diazaspiro[3,4]octan-2-yl)-N-(pyrimidin-2-ylmethyl)butanamide (2S-FNL-30)

To a stirring solution of compound (2S-FNL-29) (250 mg, 0.57 mmol) inDCM (10 mL) was added TFA (0.44 mL) under N₂ atmosphere and stirred for2 h at RT. After consumption of the starting material (by TLC), thereaction mixture was concentrated under reduced pressure. The obtainedcrude material was triturated with diethylether/n-pentane (5 mL/5 mL)and dried under reduced pressure to afford (2S-FNL-30) (180 mg, 94.7%)as semi solid (TFA salt).

¹H-NMR: (400 MHz, D₂O): δ 8.82 (d, J=2.0 Hz, 2H), 7.53 (t, J=4.8 Hz,1H), 4.67-4.62 (m, 2H), 4.44-4.40 (m, 1H), 4.34-4.32 (m, 2H), 3.61-3.56(m, 2H), 2.51-2.20 (m, 4H), 1.55-1.46 (m, 3H), 1.32-1.29 (m, 3H)

LCMS (ESI): m/z 333.3

HPLC: 90.7%

Synthesis of(2S,3R)-3-hydroxy-2-(5-isobutyryl-1-methyl-3-oxo-2,5-diazaspiro[3,4]octan-2-yl)-N-(pyrimidin-2-ylmethyl)butanamide (2S-FNL-31)

To a stirring solution of compound (2S-FNL-30) (150 mg, 0.45 mmol) inDCM (5 mL) was added TEA (0.18 mL, 1.35 mmol) followed by isobutyrylchloride (57 mg, 0.54 mmol) at 0° C. under N₂ atmosphere and stirred for2 h at RT. After consumption of the starting material (by TLC), thereaction mixture was diluted with water (5 mL) and extracted with CH₂Cl₂(2×10 mL). The combined organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure. Obtained crudematerial was purified by silica gel column chromatography eluting 2%MeOH/DCM to afford (2S-FNL-31) (85 mg, 47%) as semi solid.

¹H-NMR: (400 MHz, CD₃OD): δ 8.73 (d, J=4.8 Hz, 2H), 7.36 (t, J=4.8 Hz,1H), 4.83-4.54 (m, 3H), 4.35-4.32 (m, 1H), 4.22-4.11 (m, 1H), 3.93-3.88(m, 1H), 3.76-3.71 (m, 1H), 3.67-3.60 (m, 2H), 2.81-2.76 (m, 1H),2.21-2.07 (m, 3H), 1.96-1.91 (m, 1H), 1.29-1.26 (m, 6H), 1.05-1.02 (m,6H)

LCMS (ESI): m/z 404.4

HPLC: 93.57%

Synthesis of tert-butyl2-(((2S,3R)-3-(benzyloxy)-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)carbamoyl)-2-(1-hydroxyethyl) pyrrolidine-1-carboxylate(2S-22)

To a stirring solution of compound 2S-AN (2.5 g, 9.65 mmol) in CH₂Cl₂(50 mL) were added compound 2S-BM (2.7 g, 10.6 mmol), EDCI.HCl (2.7 g,14.4 mmol) followed by HOBt (1.9 g, 14.4 mmol) and DIPEA (5.3 mL, 28.9mmol) at 0° C. and stirred for 12 h. After consumption of the startingmaterial (by TLC), the reaction mixture was diluted with water (30 mL)and extracted with CH₂Cl₂ (2×50 mL). The separated organic layer waswashed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. Obtained crude material waspurified by silica gel column chromatography eluting 2% MeOH/DCM toafford compound 2S-22 (3.5 g, 73%) as colorless liquid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.11 (d, J=9.0 Hz, 1H), 7.82 (d, J=8.5 Hz,1H), 7.33-7.26 (m, 5H), 6.56 (s, 1H), 4.68-4.63 (m, 1H), 4.56 (s, 2H),3.80-3.74 (m, 1H), 3.55-3.33 (m, 5H), 1.76-1.66 (m, 7H), 1.40 (s, 9H),1.37-1.24 (m, 2H), 1.08-0.97 (m, 6H).

Mass (EST): m/z 504 [M⁺+1].

Synthesis of tert-butyl2-((2S,3R)-3-(benzyloxy)-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-1-methyl-3-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-23)

To a stirring solution of compound 2S-22 (3.5 g, 6.95 mmol) in THF (50mL) was added triphenylphosphine (3.6 g, 13.9 mmol) and DTAD (3.2 g,13.9 mmol). The reaction mixture was stirred at RT for 16 h. Afterconsumption of the starting material (by TLC), the reaction wasconcentrated under reduced pressure. The crude material was purified bysilica gel column chromatography eluting 30% EtOAc/hexane to affordcompound 2S-23 (1.0 g, 30%) as pale yellow liquid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.63-7.54 (m, 1H), 7.41-7.24 (m, 4H),4.60-4.37 (m, 3H), 3.98 (d, J=10.0 Hz, 1H), 3.91 (d, J=7.0 Hz, 1H), 3.77(d, J=7.0 Hz, 2H), 3.44-3.34 (m, 4H), 2.01-1.91 (m, 2H), 1.85-1.68 (m,6H), 1.40 (s, 9H), 1.20-1.11 (m, 6H).

Mass (ESI): m/z 486.6 [M⁺+1].

Synthesis of(2S,3R)-2-(5-(tert-butoxycarbonyl)-1-oxo-2,5-diazaspiro[3,4]octan-2-yl)-3-hydroxybutanoicacid (2S-24)

To a stirring solution of compound 2S-23 (1 g) in methanol (30 mL) wasadded 10% Pd/C (400 mg) at RT and stirred for 12 h under H₂ atmosphere(balloon pressure). After consumption of the starting material (by TLC),the reaction mixture was filtered through a pad of elite and the pad waswashed with methanol. Obtained filtrate was concentrated under reducedpressure to afford compound 2S-24 (230 mg, 28%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 4.79 (br s, 1H), 4.34 (br s, 1H), 4.27 (d,J=8.5 Hz, 1H), 4.03-3.95 (m, 1H), 3.78 (d, J=6.5 Hz, 1H), 3.67-3.63 (m,1H), 3.53-3.49 (m, 2H), 3.39 (t, J=9.0 Hz, 2H), 2.04-1.67 (m, 8H), 1.36(s, 9H), 1.26 (d, J=6.0 Hz, 3H), 1.08, 1.06 (dd, J=6.5 Hz, 3H).

LCMS: 396.4 [M⁺+1].

Synthesis of 2-((2S,3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-3-methyl-2,5-diazaspiro[3,4]octan-1-one (2S-FNL-32)

To a stirring solution of compound 2S-24 (230 mg, 0.58 mmol) in CH₂Cl₂(2 mL) was added TFA (0.44 mL, 5.82 mmol) at 0° C. and stirred at RT for2 h. After completion of reaction (by TLC), the reaction mixture wasconcentrated under reduced pressure to afford (2S-FNL-32) was trituratedwith pentane and diethyl ether (5 mL/5 mL) (210 mg, 92%) as sticky solid(TFA salt).

¹H-NMR: (400 MHz, D₂O): δ 4.27 (d, J=8.0 Hz, 1H), 4.07-4.03 (m, 1H),4.01-3.97 (m, 1H), 3.52-3.48 (m, 1H), 3.39-3.35 (m, 2H), 3.32-3.20 (m,3H), 2.16 (t, J=7.6 Hz, 2H), 2.06-1.96 (m, 2H), 1.89-1.80 (m, 2H),1.78-1.74 (m, 2H), 1.43 (d, J=6.4 Hz, 3H), 1.06 (d, J=6.0 Hz, 3H);

Synthesis of tert-butyl2-((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)-6-methyl-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-33)

To a stirring solution of compound 2S-BC (1.5 g, 4.38 mmol) in DCM (25mL) were added N,N-diisopropylethylamine (2.35 mL, 13.14 mmol), NH₄Cl(310 mg, 8.76 mmol), followed by EDCI (1 g, 5.25 mmol), HOBT (793 mg,5.25 mmol) at 0° C. and stirred at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was diluted with water(20 mL). The separated organic layer was washed with citric acidsolution (1×30 mL) followed by brine solution (1×30 mL). The organiclayer was dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to afford crude compound which was purified by columnchromatography by eluting 5% MeOH/DCM to obtained compound (2S-FNL-33)(600 mg, 41%) as white solid.

¹H-NMR: (400 MHz, CD₃OD): δ 4.24-4.17 (m, 1H), 4.03-3.99 (m, 3H),3.67-3.46 (m, 1H), 2.40-1.99 (m, 3H), 1.68-1.62 (m, 1H), 1.46 (s, 9H),1.24-1.18 (m, 6H);

LCMS (ESI): m/z 342.5 [M′+1]

Synthesis of(2S,3R)-3-hydroxy-2-(6-methyl-1-oxo-2,5-diazaspiro[3,4]octan-2-yl)butanamide (2S-FNL-34)

To a stirring solution of compound (2S-FNL-33) (200 mg, 0.58 mmol) inDCM (10 mL) was added trifluoroacetic acid (0.5 mL), at 0° C. under N₂atmosphere. The reaction mixture was stirred at RT for 2 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasconcentrated under reduced pressure to afford crude, which wastriturated with n-pentane (10 mL) to afford (2S-FNL-34) (100 mg, 71%) aswhite solid.

¹H-NMR: (400 MHz, D₂O): δ 4.35-4.27 (m, 2H), 4.08-3.93 (m, 3H),2.58-2.52 (m, 1H), 2.48-2.44 (m, 2H), 1.92-1.86 (m, 1H), 1.51, 1.48 (dd,J=6.8 Hz, 6.4 Hz, 3H), 1.31, 1.28 (dd, J=6.0 Hz, 6.4 Hz, 3H);

LCMS (ESI): 241.3 [M⁺+1]

Synthesis of tert-butyl2-(((2S,3R)-3-(benzyloxy)-1-oxo-1-(pyrrolidin-1-yl) butan-2-yl)carbamoyl)-2-(hydroxymethyl)-5-methylpyrrolidine-1-carboxylate (2S-25)

To a stirring solution of compound 2S-AZ (1.1 g, 4.28 mmol) in DCM (20mL) were added N,N-diisopropylethylamine (2.2 mL, 12.8 mmol), 2S-BM (1.2g, 4.78 mmol), followed by EDCI (2.45 g, 12.8 mmol), HOBT (1.7 g, 12.8mmol) at 0° C. and stirred at RT for 12 h. After consumption of thestarting material (by TLC), the reaction mixture was diluted with water(10 mL). The separated organic layer was washed with saturated NaHCO₃solution (1×25 mL), followed by brine solution (1×30 mL). The separatedorganic layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to afford crude compound which was purified by columnchromatography eluting 5% MeOH/DCM to obtained compound 2S-25 (1.2 g,57%) as a thick white syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.93 (t, J=7.6 Hz, 1H), 7.71-7.26 (m, 5H),5.30 (br s, 1H), 4.65-4.61 (m, 1H), 4.57 (s, 2H), 3.93-3.85 (m, 2H),3.57-3.34 (m, 2H), 3.17-3.09 (m, 2H), 2.07-1.94 (m, 2H), 1.77-1.73 (m,4H), 1.36-1.28 (m, 10H), 1.20 (s, 9H);

LCMS: m/z 504.7 [M⁺+1].

Synthesis of tert-butyl2-((2S,3R)-3-(benzyloxy)-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-6-methyl-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-26)

To a stirring solution of compound 2S-25 (0.6 g, 1.19 mmol) in THF (10mL) was added triphenylphosphine (0.46 g, 1.78 mmol) and DTAD (0.4 g,1.78 mmol). The reaction mixture was stirred at RT for 16 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with water (10 mL). The separated organic layer was washed withbrine solution (1×30 mL). The separated organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to afford crudecompound which was purified by column chromatography eluting 2% MeOH/DCMto obtained compound 2S-26 (0.2 g, 35%) as white solid.

¹H-NMR: (500 MHz, DMSO-d6): δ 7.63-7.26 (m, 5H), 4.52 (s, 2H), 3.91-3.77(m, 3H), 3.56-3.36 (m, 4H), 2.35-2.11 (m, 4H), 1.94-1.68 (m, 6H), 1.39(s, 9H), 1.13, 1.09 (dd, J=6.0 Hz, 5.5 Hz, 3H), 1.04 (d, J=6.5 Hz, 3H);

LCMS: m/z 486.6 [M⁺+1].

Synthesis of tert-butyl2-((2S,3R)-3-(benzyloxy)-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-6-methyl-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-27)

To a stirring solution of compound 2S-26 (1.5 g, 3.09 mmol) in methanol(20 mL) was added 10% Pd/C (200 mg) under N₂ atmosphere. The reactionmixture was stirred under H₂ atmosphere (balloon pressure) at RT for 4h. After consumption of the starting material (by TLC), the reactionmixture was filtered through a pad of celite and the pad was washed withmethanol. Obtained filtrate was concentrated under reduced pressure toafford crude compound which was purified by column chromatography toobtained compound 2S-27 (0.9 g, 90.9%) as white solid.

¹H-NMR: (500 MHz, CDCl₃): δ4.11 (d, J=7.0 Hz, 1H), 3.96-3.90 (m, 1H),3.73 (s, 2H), 3.49 (d, J=13.0 Hz, 2H), 3.40-3.34 (m, 2H), 2.50-2.28 (m,4H), 2.17-1.82 (m, 6H), 1.53 (s, 9H), 1.52-1.41 (m, 3H), 1.36-1.18 (m,3H);

LCMS: 396.5 [M⁺+1].

Synthesis of 2-((2S,3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-6-methyl-2,5-diazaspiro[3,4]octan-1-one (2S-FNL-35)

To a stirring solution of compound 2S-27 (0.18 g, 0.45 mmol) in methanol(10 mL) was added TFA (3 mL) under N₂ atmosphere at 0° C. Afterconsumption of the starting material (by TLC), the reaction mixture wasconcentrated under reduced pressure to afford crude compound which wastriturated with n-pentane (10 mL) to obtained (2S-FNL-35) (0.1 g, 74.6%)as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.99 (br s, 1H), 9.57 (br s, 1H), 4.32 (d,J=7.6 Hz, 1H), 3.93 (t, J=6.0 Hz, 1H), 3.81-3.75 (m, 3H), 3.51-3.46 (m,2H), 3.30 (t, J=6.8 Hz, 2H), 2.29-2.20 (m, 3H), 1.93-1.75 (m, 4H),1.68-1.63 (m, 1H), 1.34 (d, J=6.8 Hz, 3H), 1.13 (d, J=6.4 Hz, 3H);

Mass (ESI): m/z 296.3 [M⁺+1]

Synthesis of tert-butyl2-((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)-1,6-dimethyl-3-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-36)

To a stirring solution of compound 2S-BH (1.8 g, 5.05 mmol) in CH₂Cl₂(50 mL) were added DIPEA (2.62 mL, 15.15 mmol), EDCI (1.92 g, 10.1mmol), HOBt (1.36 g, 10.1 mmol) followed by NH₄Cl (803 mg, 15.15 mmol)at 0° C. and stirred for 12 h at RT. After consumption of the startingmaterial (by TLC), the reaction mixture was diluted with water (30 mL).The separated organic layer was washed with citric acid solution (1×50mL) followed by brine solution (1×50 mL). The organic layer was driedover anhydrous Na₂SO₄ and concentrated under reduced pressure to affordcrude compound which was purified by column chromatography by eluting 4%MeOH/DCM to afford (2S-FNL-36) (468 mg, 26%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆):δ7.25 (s, 2H), 4.92-4.48 (m, 1H), 4.34-4.01(m, 1H), 3.97-3.72 (m, 3H), 2.32-1.88 (m, 3H), 1.58-1.51 (m, 1H), 1.41(s, 9H), 1.36-1.20 (m, 6H), 1.16-1.07 (m, 3H);

LCMS (ESI): 356.4 [M⁺+1];

HPLC: 99.19%

Synthesis of(2S,3R)-2-(1,6-dimethyl-3-oxo-2,5-diazaspiro[3,4]octan-2-yl)-3-hydroxybutanamide (2S-FNL-37)

To a stirring solution of (2S-FNL-36) (200 mg, 0.56 mmol) in DCM (5 mL)was added TFA (0.45 mL, 5.63 mmol) at 0° C. and stirred at RT for 2 h.After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure. The crude material wastriturated with diethyl ether/n-pentane (50 mL/50 mL) and dried underreduced pressure to afford (2S-FNL-37) (140 mg, 98%) as hygroscopicwhite solid (TFA salt).

¹H-NMR: (400 MHz, D₂O): δ4.42-4.36 (m, 1H), 4.34-4.28 (m, 1H), 4.27-4.15(m, 1H), 4.07-4.01 (m, 1H), 2.57-2.49 (m, 1H), 2.46-2.36 (m, 2H),2.01-1.90 (m, 1H), 1.56-1.50 (m, 6H), 1.32-1.29 (m, 3H);

LCMS (ESI): 256.4 [M⁺+1];

HPLC (ELSD): 93.86%.

Synthesis oftert-butyl2-((2S,3R)-3-(benzyloxy)-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)arbamoyl)-2-(1-hydroxyethyl)-5-methylpyrrolidine-1-carboxylate (2S-28)

To a stirring solution of compound 2S-BE (2 g, 7.32 mmol) in DMF (20 mL)were added N,N-diisopropylethylamine (6.7 mL, 36.5 mmol), 2S-BM (2.6 g,8.7 mmol), followed by HATU (3.3 g, 8.7 mmol) at 0° C. and stirred at RTfor 16 h. After consumption of the starting material (by TLC), thereaction mixture was diluted with water (100 mL) and EtOAc (200 mL). Theseparated organic layer was washed with sodium bicarbonate solution(2×75 mL), citric acid solution (2×50 mL) followed by brine solution (lx50 mL). The separated organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to afford crude compound which waspurified by column chromatography by eluting 40% EtOAc/n-hexane toobtain compound 2S-28 (1 g, 27%) as pale yellow liquid;

LCMS (ESI): 518 [M⁺+1]

Synthesis of tert-butyl2-((2S,3R)-3-(benzyloxy)-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-1,6-dimethyl-3-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-29)

To a stirring solution of triphenylphosphine (1.5 g, 5.7 mmol) in THF(10 mL) was added DIAD (976 mg, 4.8 mmol) as portion-wise and stirredfor 20 min at RT. To this was added compound 2S-28 (1 g, 1.93 mmol) inTHF (10 mL) slowly at RT and stirred for 4 h. After consumption of thestarting material (by LCMS), the reaction mixture was concentrated underreduced pressure. The crude material was purified by silica gel columnchromatography eluting 30% EtOAc/hexane to afford compound 2S-29 (500mg, 63%) as yellow liquid.

¹H-NMR: (400 MHz, CDCl₃): δ7.69-7.66 (m, 1H), 7.48-7.43 (m, 1H),7.32-7.29 (m, 3H), 4.68 (s, 2H), 4.46-4.40 (m, 1H), 4.26-4.05 (m, 2H),3.97-3.91 (m, 0.5H), 3.87-3.81 (m, 0.5H), 3.58-3.53 (m, 1H), 3.40-3.32(m, 2H), 2.16-2.11 (m, 1H), 2.04-1.90 (m, 2H), 1.80-1.71 (m, 2H), 1.41(s, 9H), 1.32-1.21 (m, 10H), 1.17-1.15 (m, 3H).

LCMS (ESI): 500 [M⁺+1].

Synthesis of tert-butyl2-((2S,3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-1,6-dimethyl-3-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-30)

To a stirring solution of compound 2S-29 (200 mg, 0.40 mmol) in methanol(5 mL) was added 10% Pd/C (50 mg) under N₂ atmosphere. The reactionmixture was stirred under H₂ atmosphere at RT for 4 h. After consumptionof the starting material (by TLC), the reaction mixture was filteredthrough a pad of celite and the pad was washed with methanol (10 mL).Obtained filtrate was concentrated under reduced pressure to obtainedcrude compound, which was purified by column chromatography eluting 1%MeOH/DCM to afford compound 2S-30 (100 g, 61%) as yellow syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 4.93 (d, J=5.6 Hz, 1H), 4.26 (d, J=9.2 Hz,0.5H), 4.17 (d, J=7.2 Hz, 0.5H), 4.02-3.99 (m, 1H), 3.91-3.66 (m, 3H),3.33-3.30 (m, 1H), 3.55-3.50 (m, 1H), 3.19-3.16 (m, 1H), 2.69 (s, 1H),2.13-2.03 (m, 1H), 1.99-1.87 (m, 3H), 1.81-1.75 (m, 2H), 1.56-1.50 (m,1H), 1.39 (s, 9H), 1.19 (d, J=5.6 Hz, 3H), 1.13 (d, J=6.4 Hz, 6H).

LCMS: 410.5 [M⁺+1].

Synthesis of2-((2S,3R)-3-hydroxy-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)-3,6-dimethyl-2,5-diazaspiro[3,4]octan-1-one(2S-FNL-38)

To a stirring solution of compound 2S-30 (300 mg, 0.73 mmol) in DCM (20mL) was added TFA (418 mg, 3.66 mmol) at 0° C. under N_(a) atmosphere.The reaction mixture was stirred at RT for 4 h. After consumption of thestarting material (by TLC), the reaction mixture was evaporated underreduced pressure to afford crude, which was purified by preparative HPLCmethod to afford (2S-FNL-38) (140 mg, 46%) as thick syrup.

¹H-NMR: (400 MHz, D₂O): δ4.53-4.46 (m, 1H), 4.34-4.22 (m, 2H), 4.03 (d,J=6.4 Hz, 1H), 3.68 (s, 2H), 3.52-3.41 (m, 2H), 2.44-2.37 (m, 3H),2.03-1.94 (m, 5H), 1.56 (d, J=6.4 Hz, 6H), 1.27 (d, J=6.0 Hz, 3H).

LCMS (ESI): 310 [M⁺+1].

Synthesis of tert-butyl 2-(((S)-1,3-bis (benzyloxy)-1-oxopropan-2-yl)carbamoyl)-2-(1-hydroxyethyl)-5-methylpyrrolidine-1-carboxylate (2S-BN)

To a stirring solution of 2S-BE (3 g, 10.98 mmol) in DCM (30 mL) wereadded N,N-diisopropylethylamine (5.73 mL, 32.96 mmol), 2S-AC (3.75 g,13.17 mmol) followed by HATU (5 g, 13.17 mmol) at 0° C. and stirred atRT for 16 h. After consumption of the starting material (by TLC), thereaction mixture was diluted with water (20 mL). The separated organiclayer was washed with brine solution (30 mL). The organic layer wasdried over anhydrous Na₂SO₄ and concentrated under reduced pressure toafford crude compound which was purified by column chromatography byeluting 20% EtOAc/n-hexane to obtained compound 2S-BN (2.9 g, 49%) asbrown thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.50 (m, 1H), 7.33-7.27 (m, 10H),5.68-5.60 (m, 1H), 5.22-5.09 (m, 2H), 4.72-4.43 (m, 3H), 3.89-3.63 (m,3H), 2.28-1.78 (m, 3H), 1.45-1.42 (m, 1H), 1.36 (s, 9H), 1.26-1.04 (m,6H);

LCMS (ESI): m/z 541.6 [M⁺+1]

Synthesis of tert-butyl2-((S)-1,3-bis(benzyloxy)-1-oxopropan-2-yl)-1,6-dimethyl-3-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-BO)

To a stirring solution of triphenylphosphine (3.51 g, 13.42 mmol) in dryTHF (30 mL) was added DIAD (2.21 g, 10.74 mmol) as portionwise andstirred for 15 min at RT. To this precipitated solution added 2S-BN (2.9g, 5.37 mmol) in dry THF (15 mL) slowly at RT and stirred for 16 h.After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure. The crude material wastriturated with 30% di ethylether/n-pentane. The filterate wasconcentrated under reduced pressure to obtained crude compound which waspurified by silica gel column chromatography eluting 30% EtOAc/hexane toafford 2S-BO (2.5 g, 89.2%) as brown thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.38-7.25 (m, 10H), 5.22-5.15 (m, 2H),4.80-4.73 (m, 2H), 4.56-4.43 (m, 2H), 3.92-3.60 (m, 3H), 1.89-1.83 (m,3H), 1.50-1.44 (m, 1H), 1.40 (s, 9H), 1.22-1.18 (s, 3H), 1.16-1.13 (m,3H);

LCMS (ESI): m/z 523.6 [M⁺+1]

Synthesis of(2S)-2-(5-(tert-butoxycarbonyl)-1,6-dimethyl-3-oxo-2,5-diazaspiro[3,4]octan-2-yl)-3-hydroxypropanoicacid (2S-BP)

To a stirring solution of 2S-BO (2.5 g, 4.78 mmol) in methanol (50 mL)was added 10% Pd/C (800 mg) under N₂ atmosphere. The reaction mixturewas stirred under H₂ atmosphere at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was filtered through apad of celite and the pad was washed with methanol (30 mL). Obtainedfiltrate was concentrated under reduced pressure to afford crude whichwas triturated with n-pentane (30 mL) to afford 2S-BP (900 mg, 56.2%) assticky solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 4.78-4.75 (m, 1H), 4.24-4.18 (m, 1H),3.86-3.81 (m, 1H), 3.80-3.72 (m, 2H), 3.64-3.59 (m, 1H), 2.15-1.93 (m,3H), 1.55-1.50 (m, 1H), 1.39 (s, 9H), 1.24-1.10 (m, 6H);

LCMS (ESI): m/z 343.3 [M⁺+1]

Synthesis of tert-butyl2-((S)-3-hydroxy-1-(isopropylamino)-1-oxopropan-2-yl)-1-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-39)

To a stirring solution of 2S-AF (200 mg, 0.63 mmol) in CH₂Cl₂ (10 mL)were added DIPEA (0.32 mL, 1.90 mmol), isopropyl amine (0.08 mL, 0.94mmol), HATU (287 mg, 0.75 mmol) at 0° C. and stirred to RT for 5 h.After consumption of the starting material (by TLC), the reactionmixture was diluted with water (10 mL). The separated organic layer waswashed with citric acid (1×20 mL) followed by brine solution (1×20 mL).The organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. Obtained crude material waspurified by preparative HPLC purification to afford (2S-FNL-39) (150 mg,67.2%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.76 (d, J=8.4 Hz, 1H), 5.01-4.91 (m, 1H),4.70 (t, J=6.0 Hz, 1H), 4.14-4.07 (m, 1H), 3.99-3.80 (m, 2H), 3.78-3.61(m, 2H), 3.58-3.35 (m, 2H), 2.20-2.05 (m, 2H), 1.85-1.77 (m, 2H), 1.43(s, 9H), 1.10-1.00 (m, 6H)

Mass (ESI): m/z 356.6 [M⁺+1]

HPLC: 99.27%

Synthesis of tert-butyl2-((2S,3R)-1-(benzylamino)-3-hydroxy-1-oxobutan-2-yl)-1,6-dimethyl-3-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-40)

To a stirring solution of 2S-BH (250 mg, 0.70 mmol) in CH₂Cl₂ (10 mL)were added DIPEA (0.36 mL, 2.11 mmol), EDCI (161 mg, 0.84 mmol), HOBt(129 mg, 0.84 mmol) followed by benzylamine (82 mg, 0.77 mmol) at 0° C.and stirred for 12 h at RT. After consumption of the starting material(by TLC), the reaction mixture was diluted with water (10 mL). Theseparated organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to afford crude compound which was purified bycolumn chromatography by eluting 5% MeOH/DCM to afford (2S-FNL-40) (55mg, 16%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.31-7.20 (m, 5H), 4.92-4.87 (m, 1H),4.64-4.55 (m, 1H), 4.46-4.37 (m, 2H), 4.22-4.10 (m, 1H), 4.02-3.90 (m,2H), 2.39-1.95 (m, 3H), 1.70-1.60 (m, 1H), 1.37 (s, 9H), 1.30-1.22 (m,9H);

LCMS (ESI): m/z 446.56 [M⁺+1];

HPLC: 89.54%

Synthesis of tert-butyl2-((2S,3R)-1-((4-fluorobenzyl)amino)-3-hydroxy-1-oxobutan-2-yl)-1,6-dimethyl-3-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-41)

To a stirring solution of 2S-BH (500 mg, 1.40 mmol) in CH₂Cl₂ (10 mL)were added DIPEA (0.73 mL, 4.21 mmol), EDCI (321 mg, 1.68 mmol), HOBt(257 mg, 1.68 mmol) followed by 4-fluoro benzylamine (175 mg, 1.40 mmol)at 0° C. and stirred for 12 h at RT. After consumption of the startingmaterial (by TLC), the reaction mixture was diluted with water (15 mL).The separated organic layer was washed with citric acid solution (1×25mL) followed by brine solution (1×25 mL). The organic layer was driedover anhydrous Na₂SO₄ and concentrated under reduced pressure to affordcrude compound which was purified by column chromatography by eluting 5%MeOH/DCM followed by preparative HPLC purification to afford (2S-FNL-41)(150 mg, 23.07%) as white solid.

¹H-NMR: (400 MHz, CD₃OD): δ 7.34-7.30 (m, 2H), 7.02-6.98 (m, 2H),4.65-4.59 (m, 1H), 4.55-4.36 (m, 2H), 4.34-4.20 (m, 1H), 4.12-3.99 (m,2H), 2.39-2.31 (m, 1H), 2.19-2.01 (m, 2H), 1.71-1.62 (m, 1H), 1.40 (s,9H), 1.29-1.13 (m, 9H);

LCMS (ESI): m/z 464.5 [M⁺+1];

HPLC: 96.32%

Synthesis of tert-butyl2-((2S,3R)-3-hydroxy-1-((4-methoxybenzyl)amino)-1-oxobutan-2-yl)-1,6-dimethyl-3-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-42)

To a stirring solution of 2S-BH (250 mg, 0.70 mmol) in CH₂Cl₂ (10 mL)were added DIPEA (0.36 mL, 2.11 mmol), EDCI (161 mg, 0.84 mmol), HOBt(129 mg, 0.84 mmol) followed by 4-methoxy benzylamine (106 mg, 0.77mmol) at 0° C. and stirred for 12 h at RT. After consumption of thestarting material (by TLC), the reaction mixture was diluted with water(10 mL). The separated organic layer was washed with citric acidsolution (1×20 mL) followed by brine solution (1×25 mL). The organiclayer was dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to afford crude compound which was purified by columnchromatography by eluting 5% MeOH/DCM to afford (2S-FNL-42) (60 mg,17.9%) as an off-white solid.

¹H-NMR: (400 MHz, CD₃OD): δ 7.24 (d, J=1.6 Hz, 2H), 6.85 (d, J=1.6 Hz,2H), 4.64-4.58 (m, 1H), 4.39-4.28 (m, 1H), 4.21-4.08 (m, 2H), 4.06-3.99(m, 1H), 3.98-3.88 (m, 1H), 3.83 (s, 3H), 2.39-2.28 (m, 1H), 2.22-2.13(m, 1H), 2.09-1.97 (m, 1H), 1.71-1.61 (m, 1H), 1.40 (s, 9H), 1.31-1.22(m, 9H);

LCMS (ESI): m/z 476.6 [M⁺+1];

HPLC: 90.29%

Synthesis of tert-butyl2-((2S,3R)-3-hydroxy-1-(isopropylamino)-1-oxobutan-2-yl)-1,6-dimethyl-3-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-43)

To a stirring solution of 2S-BH (500 mg, 1.40 mmol) in CH₂Cl₂ (10 mL)were added DIPEA (0.73 mL, 4.21 mmol), isopropyl amine (100 mg, 1.68mmol), HATU (798 mg, 2.1 mmol) at 0° C. and stirred for 12 h at RT.After consumption of the starting material (by TLC), the reactionmixture was diluted with water (10 mL). The separated organic layer waswashed with citric acid solution (15 mL) followed by brine solution (15mL). The organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to afford crude compound which was purified bycolumn chromatography by eluting 2% MeOH/DCM followed by preparativeHPLC purification to afford (2S-FNL-43) (100 mg, 18%) as white solid.

¹H-NMR: (400 MHz, D₂O): δ 4.42-3.89 (m, 5H), 2.38-2.04 (m, 3H),1.77-1.72 (m, 1H), 1.40 (s, 9H), 1.36-1.17 (m, 15H)

LCMS (ESI): m/z 398.5 [M⁺+1];

HPLC: 93.36%

Synthesis of tert-butyl2-((2S,3R)-1-(tert-butylamino)-3-hydroxy-1-oxobutan-2-yl)-1,6-dimethyl-3-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-44)

To a stirring solution of 2S-BH (500 mg, 1.40 mmol) in CH₂Cl₂ (10 mL)were added DIPEA (0.62 mL, 3.51 mmol), tert-butyl amine (125 mg, 1.68mmol), HATU (798 mg, 2.1 mmol) at 0° C. and stirred for 12 h at RT.After consumption of the starting material (by TLC), the reactionmixture was diluted with water (10 mL). The separated organic layer waswashed with citric acid solution (15 mL) followed by brine solution (15mL). The organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to afford crude compound which was purified bycolumn chromatography by eluting 2% MeOH/DCM followed by preparativeHPLC purification to afford (2S-FNL-44) (100 mg, 17.3%) as white solid.

¹H-NMR: (500 MHz, CD₃OD): δ 4.53-4.50 (m, 1H), 4.08-3.99 (m, 2H),3.82-3.79 (m, 1H), 2.38-2.34 (m, 1H), 2.20-2.17 (m, 2H), 2.09-2.01 (m,1H), 1.71-1.67 (m, 1H), 1.40 (s, 9H), 1.38 (s, 9H), 1.33-1.21 (m, 9H);

LCMS (ESI): m/z 412.5 [M⁺+1];

HPLC: 93.91%

Synthesis of tert-butyl 2-((S)-1-((4-fluorobenzyl)amino)-3-hydroxy-1-oxopropan-2-yl)-1,6-dimethyl-3-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-45)

To a stirring solution of 2S-BP (200 mg, 0.58 mmol) in DCM (10 mL) wereadded N,N-diisopropylethylamine (0.3 mL, 1.75 mmol), EDCI (133 mg, 0.69mmol), HOBT (93 mg, 0.69 mmol) followed by 4-fluoro benzylamine (79.7mg, 0.63 mmol) at 0° C. and stirred at RT for 16 h. After consumption ofthe starting material (by TLC), the reaction mixture was diluted withwater (20 mL). The separated organic layer was washed with citric acid(20 mL) followed by brine solution (30 mL). The separated organic layerwas dried over anhydrous Na₂SO₄ and concentrated under reduced pressureto afford crude compound which was purified by column chromatography byeluting 3% MeOH/DCM to obtained (2S-FNL-45) (46 mg, 17.7%) as thicksyrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.63-8.59 (m, 1H), 7.30-7.26 (m, 2H),7.15-7.07 (m, 2H), 5.07-5.00 (m, 1H), 4.31-4.21 (m, 3H), 3.89-3.62 (m,4H), 2.13-1.84 (m, 3H), 1.58-1.52 (m, 1H), 1.36 (s, 9H), 1.32-1.20 (m,3H), 1.18-1.13 (m, 3H);

LCMS (ESI): m/z 450.5 [M⁺+1]

HPLC: 93%

Synthesis of tert-butyl 2-((S)-1-((4-fluorobenzyl)amino)-3-hydroxy-1-oxopropan-2-yl)-1,6-dimethyl-3-oxo-2,5-diazaspiro[3,4]octane-5-carboxylate(2S-FNL-46)

To a stirring solution of 2S-BP (500 mg, 1.46 mmol) in DCM (15 mL) wereadded N,N-diisopropylethylamine (0.76 mL, 4.38 mmol), cyclobutylamine(124 mg, 1.75 mmol) followed by HATU (665 mg, 1.75 mmol) at 0° C. andstirred at RT for 16 h. After consumption of the starting material (byTLC), the reaction mixture was diluted with water (20 mL). The separatedorganic layer was washed with citric acid (20 mL) followed by brinesolution (30 mL). The organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure to afford crudecompound which was purified by column chromatography by eluting 3%MeOH/DCM to obtained (2S-FNL-46) (110 mg, 19%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.23 (d, J=8.0 Hz, 1H), 4.98-4.83 (m, 1H),4.30-4.13 (m, 2H), 3.95-3.76 (m, 2H), 3.72-3.66 (m, 2H), 2.49-1.89 (m,3H), 1.64-1.54 (m, 3H), 1.48 (s, 9H), 1.19-1.12 (m, 10H);

LCMS (ESI): m/z 396.5 [M⁺+1]

HPLC: 96.6%

Example 3—[³H] MK-801 Binding Assay

Methods

Assays were conducted as described in Moskal et al. (Moskal, J. R., Kuo,A. G., Weiss, C., Wood, P. L., O'Connor Hanson, A., Kelso, S., Harris,R. B., Disterhoft, J. F., 2005. GLYX-13: a monoclonal antibody-derivedpeptide that acts as an N-methyl-D-aspartate receptor modulator.Neuropharmacology. 49, 1077-87) The potentiation of [³H]MK-801 binding(5 nM; 22.5 Ci/mmol) to well washed rat cortical membranes (200 μg) wasmeasured under non-equilibrium conditions (15 min @ 25° C.) in thepresence of increasing concentrations of test compounds and 50 μMglutamate. Zero levels were determined in the absence of any glycineligand and in the presence of 30 μM 5,7 DCKA. Maximal stimulation wasmeasured in the presence of 1 mM glycine, and 50 μM glutamate waspresent in all samples. The facilitation of [³H]MK-801 binding by testscompounds was calculated by using a 3 parameter log agonist vs. responseequation (Graph pad Prism, USA) and potency (EC₅₀, expressed in pM) andmaximal activity (% maximal stimulation) were calculated for the testcompound.

Results

As shown in Table 2 and FIG. 1, the pEC50 and maximal activity forCompound X are −7.4 and 38%.

TABLE 2 Activity Compound pEC50 (%) X −7.4 38

TABLE 3 Additional Biological Data Unified Unified Unified UnifiedActivity Activity Unified Activity Activity Data: LTP, Data: PorsoltUnified Unified Activity [3H] MK-801 Data: LTP Data: LTP Significant (S)Floating Time Activity Activity Data: Porsolt binding assay:Augmentation Concentration or Non-significant Inhibition Data: PorsoltData: Porsolt Time Post Compound EC50 (M) (Percent) (uM) (NS) (Percent)Dose (mg/kg) Dose, route Dose (Hours) 25-FNL-3 5.43E−08 130 1 S 80 3 IV1 25-FNL-21 80 0.1 NS 25-FNL-7  1.1E−08 25-FNL-27 3.49E−12 25-FNL-34 1000.1 S 73 1 PO 1

Example 4—Long Term Potentiation in Hippocampal Slices

Methods

Assays were conducted as described in Zhang et al. (Zhang, X. L.,Sullivan, J. A., Moskal, J. R., Stanton, P. K., 2008. A NMDA receptorglycine site partial agonist, GLYX-13, simultaneously enhances LTP andreduces LTD at Schaffer collateral-CA1 synapses in hippocampus.Neuropharmacology. 55, 1238-50) Sprague-Dawley rats (12-18 days old;Taconic Farms) were deeply anesthetized with isoflurane and decapitated.Rat brains were removed rapidly, submerged in ice-cold artificialcerebrospinal fluid (ACSF, 2-4° C.), which contained (in mM): 124 NaCl,4 KCl, 2 MgSO4, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose; at pH 7.4,gassed continuously with 95% O2/5% CO2). The rat brains were hemisected,the frontal lobes cut off, and individual hemispheres glued usingcyanoacrylate adhesive onto a stage immersed in ice-cold ACSF gassedcontinuously with 95% O2/5% CO2 during slicing. Coronal slices (400 μmthick) were cut using a Vibratome (Leica VT1200S), and transferred to aninterface holding chamber for incubation at room temperature for aminimum of one hour before transferring to a Haas-style interfacerecording chamber continuously perfused at 3 ml/min with oxygenated ACSFat 32±0.5° C. Low resistance recording electrodes were made fromthin-walled borosilicate glass (1-2 MΩ after filling with ACSF) andinserted into the apical dendritic region of the Schaffer collateraltermination field in stratum radiatum of field CA1 region to recordfield excitatory postsynaptic potentials (fEPSPs). A bipolar stainlesssteel stimulating electrode (FHC Co.) was placed on Schaffercollateral-commissural fibers in CA3 stratum radiatum, and constantcurrent stimulus intensity adjusted to evoke approximately half-maximalfEPSPs once each 30 s (50-100 pA; 100 duration). fEPSP slope wasmeasured before and after induction of LTP by linear interpolation from20 to 80% of maximum negative deflection, and slopes confirmed to bestable to within ±10% for at least 15 min before commencing anexperiment. Bath application of the test compound (1 μM) was applied 30min prior to application of Schaffer collateral stimulus trains toelicit LTP. LTP was induced by stimulation of Schaffer collateral axonswith four high frequency theta burst stimulus trains of 10×100 Hz/5pulse bursts each, applied at an inter-burst interval of 200 ms. Eachtrain was 2 seconds in duration, and trains were applied 15 secondsapart. The signals were recorded using a Multiclamp 700B amplifier anddigitized with a Digidata 1322 (Axon Instruments, USA). Data wereanalyzed using pClamp software (version 9, Axon Instruments) on anIBM-compatible personal computer.

Results

As shown in FIG. 2, Compound X tested at 1 μM increased long-termpotentiation after high frequency stimulation of rat Schaffercollateral-evoked NMDA e.p.s.c.s recorded in CA1 pyramidal neurons.

TABLE 4 Additional Biological Data MK-801 Glycine Site Binding LTP: LTPAssay: LTP: LTP LTP: LTP Significance, Rat Cortex AugmentationConcentration S or Compound EC50 (M) (%) (uM) NS 25-FNL-38 3.313E−0925-FNL-2  2.002E−08 25-FNL-10 1.188E−12  90 1 NS 25-FNL-14 6.133E−11 1201 NS 25-FNL-33  1.89E−08 140 1 S

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

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1.-51. (canceled)
 52. A compound represented by:

or a pharmaceutically acceptable salt thereof.
 53. A pharmaceuticalcomposition, the pharmaceutical composition comprising: a compoundrepresented by:

or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable excipient.
 54. A method of treating neuropathic pain, themethod comprising: administering to a patient in need thereof atherapeutically effective amount of a compound represented by:

or a pharmaceutically acceptable salt thereof.
 55. A method of treatingneuropathic pain, comprising: administering to a patient in need thereofa therapeutically effective amount of a pharmaceutical composition,comprising a compound represented by:

or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable excipient.